Compounds and methods for regulating cell adhesion

ABSTRACT

Methods for using modulating agents to enhance or inhibit cadherin-mediated cell adhesion in a variety of in vivo and in vitro contexts are provided. In particular, the modulating agents may be used in the therapy of multiple sclerosis and other demyelinating diseases. The modulating agents comprise at least one cadherin cell adhesion recognition sequence (HAV) or an antibody or fragment thereof that specifically binds to a cadherin cell adhesion recognition sequence. Modulating agents may additionally comprise one or more cell adhesion recognition sequences recognized by other adhesion molecules. Such modulating agents may, but need not, be linked to a targeting agent, drug and/or support material.

TECHNICAL FIELD

The present invention relates generally to methods for modulatingcadherin-mediated processes, and more particularly to the use ofmodulating agents comprising a cadherin cell adhesion recognitionsequence, or an antibody that specifically recognizes such a sequence,for inhibiting or enhancing functions such as cell adhesion.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is a chronic neurological disease that affectsapproximately 250,000 individuals in the United States. In a patientafflicted with MS, axons become demyelinated and oligodendrocytes die.Although the clinical course can vary, the most common form ismanifested by relapsing neurological deficits, including paralysis,sensory deficits, and visual problems.

In MS and other demyelinating diseases, Schwann cells are generallyexcluded from areas of demyelination and, following axon damage,regeneration generally fails at Schwann cell-astrocyte boundaries(Carlstedt et al., Brain Res. Bulletin 22:93-102, 1989). Inhibition ofSchwann cell migration and boundary formation by astrocytes appears toplay a significant part in limiting spontaneous repair processes in thedamaged central nervous system (CNS).

In theory, Schwann cells from the peripheral nervous system could beused to replace damaged oligodendrocytes in the CNS. However, theefficacy of such treatment has been limited by poor Schwann cellmigration and by boundary formation. When Schwann cells are grafted intothe adult CNS, they can migrate along blood vessels and meningealsurfaces, but form boundaries where they meet astrocytes. Theseboundaries can present an obstacle for regenerating axons. Thus,recruitment of regenerating axons into Schwann cell grafts is frequentlypoor, and axons remaining in the grafts fail to grow back into CNStissue unless their target neurons are immediately adjacent (Brecknellet al., Neurosci. 74:775-784, 1996; Liuzzi and Lasak, Science237:642-645, 1987). Transplanted Schwann cells have been found to becapable of remyelinating central axons of normal (Blakemore, Nature266:68-69, 1977) or myelin deficient rats (Duncan et al., J. Neurocytol17:351-360, 1988), but in both of these cases the area of remyelinationis limited to the region close to the transplantation site.

Other approaches to developing a definitive treatment for MS have alsobeen largely unsuccessful. Corticosteroids and ACTH may hasten recoveryfrom acute exacerbations, but they do not prevent future attacks, thedevelopment of additional disabilities or chronic progression of MS. Inaddition, the substantial side effects of steroid treatments make thesedrugs undesirable for long-term use. Other toxic compounds, such asazathioprine, a purine antagonist, cyclophosphamide and cyclosporinehave also been used to treat symptoms of MS. Like corticosteroids,however, these drugs are beneficial at most for a short term and arehighly toxic. More recently, cytokines such as IFN-γ and IFN-β have beenadministered in attempts to alleviate the symptoms of MS, but suchtreatment has led to a clinical exacerbation for some patients.Betaseron has also been employed, but with no effect on the rate ofclinical deterioration, and side effects were commonly observed.

Accordingly, there is a need in the art for methods for treating MS thatare effective and are not associated with the disadvantages of thepresent treatments. The present invention fulfills this need and furherprovides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides methods for modulating cadherin-mediatedcell adhesion. Within one aspect, methods are provided for treating ademyelinating neurological disease, such as multiple sclerosis, in amammal, comprising administering to a mammal a cell adhesion modulatingagent that inhibits cadherin-mediated cell adhesion. The modulatingagent may comprise the sequence His-Ala-Val or may comprise an antibodyor fragment thereof that specifically binds to a cadherin cell adhesionrecognition sequence. A modulating agent may be administered byimplantation with Schwann cells or oligodendrocyte progenitor cellsand/or may be administered within a pharmaceutical composition.

Within further aspects, the present invention provides methods forreducing unwanted cellular adhesion in a mammal, comprisingadministering to a mammal a cell adhesion modulating agent that inhibitsunwanted cadherin-mediated cell adhesion resulting from surgery, injury,disease or inflammation. The modulating agent may comprise the sequenceHis-Ala-Val or may comprise an antibody or fragment thereof thatspecifically binds to a cadherin cell adhesion recognition sequence.

The present invention further provides methods for enhancing thedelivery of a drug through the skin of a mammal, comprising contactingepithelial cells of a mammal with a cell adhesion modulating agent thatinhibits cadherin-mediated cell adhesion and a drug, wherein the step ofcontacting is performed under conditions and for a time sufficient toallow passage of said dnug across said epithelial cells. The modulatingagent may comprise the sequence His-Ala-Val or may comprise an antibodyor fragment thereof that specifically binds to a cadherin cell adhesionrecognition sequence.

Within further aspects, methods are provided for enhancing the deliveryof a drug to a tumor in a mammal, comprising administering to a mammal acell adhesion modulating agent that inhibits cadherin-mediated celladhesion and a drug. The modulating agent may comprise 3-16 amino acidresidues including the sequence His-Ala-Val or may comprise an antibodyor fragment thereof that specifically binds to a cadherin cell adhesionrecognition sequence.

In a related aspect, the present invention provides methods for treatingcancer in a mammal, comprising administering to a mammal a cell adhesionmodulating agent that inhibits cadherin-mediated cell adhesion and adrug. The modulating agent may comprise 3-16 amino acid residuesincluding the sequence His-Ala-Val or may comprise an antibody orfragment thereof that specifically binds to a cadherin cell adhesionrecognition sequence.

Within furter aspects, methods are provided for inhibiting angiogenesisin a mammal, comprising administering to a mammal a cell adhesionmodulating agent that inhibits cadherin-mediated cell adhesion. Themodulating agent may comprise the sequence His-Ala-Val or may comprisean antibody or fragment thereof that specifically binds to a cadherincell adhesion recognition sequence.

The present invention further provides methods for enhancing drugdelivery to the CNS of a mammal, comprising administering to a mammal acell adhesion modulating agent that inhibits cadherin-mediated celladhesion. The modulating agent may comprise 3-16 amino acid residuesincluding the sequence His-Ala-Val or may comprise an antibody orfragment thereof that specifically binds to a cadherin cell adhesionrecognition sequence.

Within further aspects, the present invention provides methods forenhancing wound healing in a mammal, comprising contacting a wound in amammal with a cell adhesion modulating agent that enhancescadherin-mediated cell adhesion. The modulating agent may comprise thesequence His-Ala-Val or may comprise an antibody or fragment thereofthat specifically binds to a cadherin cell adhesion recognitionsequence.

In a related aspect, methods are provided for enhancing adhesion offoreign tissue implanted within a mammal, comprising contacting a siteof implantation of foreign tissue in a mammal with a cell adhesionmodulating agent that enhances cadherin-mediated cell adhesion. Themodulating agent may comprise the sequence His-Ala-Val or may comprisean antibody or fragment thereof that specifically binds to a cadherincell adhesion recognition sequence.

Within further aspects, the present invention provides methods forinducing apoptosis in a cadherin-expressing cell, comprising contactinga cadherin-expressing cell with a cell adhesion modulating agent thatinhibits cadherin-mediated cell adhesion. The modulating agent maycomprise the sequence His-Ala-Val or may comprise an antibody orfragment thereof that specifically binds to a cadherin cell adhesionrecognition sequence.

The present invention further provides methods for modulating the immunesystem of a mammal, comprising administering to a mammal apharmaceutical composition comprising a cell adhesion modulating agentthat inhibits cadherin-mediated cell adhesion. The modulating agent maycomprise the sequence His-Ala-Val or may comprise an antibody orfragment thereof that specifically binds to a cadherin cell adhesionrecognition sequence.

Within another aspect, the present invention provides methods forpreventing pregnancy in a mammal, comprising administering to a mammal acell adhesion modulating agent that inhibits cadherin-mediated celladhesion. The modulating agent may comprise the sequence His-Ala-Val ormay comprise an antibody or fragment thereof that specifically binds toa cadherin cell adhesion recognition sequence.

In still rther aspects, the present invention provides methods forincreasing vasopermeability in a mammal, comprising administering to amammal a cell adhesion modulating agent that inhibits cadherin-mediatedcell adhesion. The modulating agent may comprise the sequenceHis-Ala-Val or may comprise an antibody or fragment thereof thatspecifically binds to a cadherin cell adhesion recognition sequence.

The present invention also provides, within firther aspects, methods forenhancing and/or directing neurite outgrowth, comprising contacting aneuron with a cell adhesion modulating agent that enhancescadherin-mediated cell adhesion. The modulating agent may comprise thesequence His-Ala-Val or may comprise an antibody or fragment thereofthat specifically binds to a cadherin cell adhesion recognitionsequence.

Within related aspects, methods are provided for treating spinal cordinjuries in a mammal, comprising administering to a mammal a celladhesion modulating agent that enhances cadherin-mediated cell adhesion.The modulating agent may comprise the sequence His-Ala-Val or maycomprise an antibody or fragment thereof that specifically binds to acadherin cell adhesion recognition sequence.

The present invention also provides methods for inhibiting synapticstability in a mammal, comprising administering to a mammal a celladhesion modulating agent that inhibits cadherin-mediated cell adhesion.The modulating agent may comprise the sequence His-Ala-Val or maycomprise an antibody or fragment thereof that specifically binds to acadherin cell adhesion recognition sequence.

In still further aspects, methods are provided for identifying an agentcapable of modulating cadherin-mediated cell adhesion. One such methodcomprises the steps of: (a) contacting Schwann cells with an astrocyticsurface in the presence of candidate modulating agent; (b) washing theastrocytic surface to remove non-attached cells; and (c) comparing thenumber of Schwann cells attached to the astrocytic surface with thenumber of Schwann cells attached to an astrocytic surface in the absenceof candidate modulating agent. Another method for identifying an agentcapable of modulating cadherin-mediated cell adhesion comprises thesteps of: (a) contacting Schwann cells with polylysine- and/orlaminin-coated surface in the presence of candidate modulating agent;(b) washing the surface to remove non-attached cells; (c) contactingattached Schwann cells with an astrocyte-coated surface; and (d)comparing the migration of the attached Schwann cells with the migrationin the absence of candidate modulating agent.

A further method for identifying an agent capable of modulatingcadherin-mediated cell adhesion comprises the steps of: (a) culturingneurons on a monolayer of cells that express N-cadherin in the presenceand absence of a candidate agent, under conditions and for a timesufficient to allow neurite outgrowth, wherein the cells are transfectedwith a polynucleotide encoding N-cadherin and wherein the cells do notexpress a detectable level of N-cadherin in the absence of transfectionwith such a polynucleotide; (b) determining a mean neurite length forthe neurons; and (c) comparing the mean neurite length for neuronscultured in the presence of candidate agent to the neurite length forneurons cultured in the absence of candidate agent.

Yet another method for identifying an agent capable of modulatingcadherin-mediated cell adhesion comprises the steps of: (a) culturingcells that express a cadherin in the presence and absence of a candidateagent, under conditions and for a time sufficient to allow celladhesion; and (b) visually evaluating the extent of cell adhesion amongthe cells.

A further method for identifiing an agent capable of modulatingcadherin-mediated cell adhesion comprises the steps of: (a) culturingnormal rat kidney (NRK) cells in the presence and absence of a candidateagent, under conditions and for a time sufficient to allow celladhesion; and (b) comparing the level of cell surface E-cadherin forcells cultured in the presence of candidate agent to the level for cellscultured in the absence of candidate agent.

Another method for identifying an agent capable of modulatingcadherin-mediated cell adhesion comprises the steps of: (a) contactingan epithelial surface of skin with a test marker in the presence andabsence of candidate agent; and (b) comparing the amount of test markerthat passes through the skin in the presence of candidate agent to theamount that passes through skin in the absence of candidate agent.

Within further aspects, the present invention provides methods fordetecting the presence of cadherin-expressing cells in a sample,comprising: (a) contacting a sample with an antibody that binds to amodulating agent comprising the sequence His-Ala-Val under conditionsand for a time sufficient to allow formation of an antibody-cadherincomplex; and (b) detecting the level of antibody-cadherin complex.

Within a related aspect, the present invention provides kits fordetecting the presence of cadherin-expressing cells in a sample,comprising: (a) an antibody that binds to a modulating agent comprisingthe sequence His-Ala-Val; and (b) a detection reagent.

The present invention also provides, within a further aspect, kits forenhancing transdermal drug delivery, comprising: (a) a skin patch; and(b) a cell adhesion modulating agent, wherein the modulating agentcomprises the sequence His-Ala-Val, and wherein the modulating agentinhibits cadherin-mediated cell adhesion.

Within another aspect, the present invention provides methods foridentifying a compound capable of modulating cadherin-mediated celladhesion, comprising: (a) contacting an antibody that binds to amodulating agent comprising the sequence His-Ala-Val with a testcompound; and (b) detecting the level of antibody that binds to the testcompound, and therefrom identifyig a compound capable of modulatingcadherin-mediated cell adhesion.

These and other aspects of the invention will become evident uponreference to the following detailed description and attached drawings.All references disclosed herein are hereby incorporated by reference intheir entirety as if each were individually noted for incorporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting the structure of classical cadherins. Thefive extracellular domains are designated EC1-EC5, the hydrophobicdomain that transverses the plasma membrane (PM) is represented by TM,and the two cytoplasmic domains are represented by CP1 and CP2. Thecalcium binding motifs are shown by DXNDN (SEQ ID NO: 1), DXD and LDRE(SEQ ID NO. 2). The cell adhesion recognition (CAR) sequence, HAV, isshown within EC1. Cytoplasmic proteins β-catenin (β), α-catenin (α) andα-actinin (ACT), which mediate the interaction between cadherins andmicrofilaments (MF) are also shown.

FIG. 2 provides the amino acid sequences of mammalian classical cadherinEC1 domains: human N-cadherin (SEQ ID NO: 3), mouse N-cadherin (SEQ IDNO: 4), cow N-cadherin (SEQ ID NO: 5), human P-cadherin (SEQ ID NO: 6),mouse P-cadherin (SEQ ID NO: 7), human E-cadherin (SEQ ID NO: 8) andmouse E-cadherin (SEQ ID NO: 9).

FIGS. 3A-3D are photographs showing cellular boundaries generated inculture. FIGS. 3A and 3C represent Schwann cell-astrocyte boundaries.FIG. 3D is the fluorescent counterpart of 3C. Astrocytes (green) arestained with anti-GFAP and Schwann cells (orange) are stained with antiGAP-43. Notice the Schwann cells surrounding one lone astrocyte and onlyoccasional “finger-like” processes of Schwann cells overlapping theastrocytes. FIG. 3B depicts a typical Schwann cell-skin fibroblast“boundary”; note the lack of a defined territorial margin with clearcellular overlap. Scale bar 50 μm.

FIGS. 4A and 4B are graphs depicting Schwann cell migration upon avariety of cellular substrates as determined by the inverted coverslipmigration assay. 1 mm×2 mm glass fragments laden with fluorescentlylabeled Schwann cells were inverted onto various substrates and left for2 days. The number of migrated Schwann cells per unit distance wasrecorded and is shown as FIG. 4A; the furthest distance of migration bythe Schwann cells from the edge of the fragment is shown as FIG. 4B.Schwann cells migrated poorly upon primary astroglial cultures and uponthe astrocytic cell line A7 compared to fibroblastic or meningeal cellsubstrates. The most migration was found upon the non-cellularsubstrate, laminin. All data are expressed as the mean±S.E.C. of atleast three separate determinations; each determination consisting of atleast 8-12 fragments per group. One way analysis of variance performedupon the maximum migratory distances revealed a statistical difference<0.001 between at least one of the groups. A post hoc multiplecomparisons test (Tukey test) revealed significant differences (p<0.01)between the groups marked with (*) and the control astrocytes.

FIGS. 5A and 5B are graphs depicting the migration of Schwann cells onastrocyte matrix as determined by the inverted-fragmented-coverslipmigration assay. 1 mm×2 mm glass fragments laden with fluorescentlylabeled Schwann cells were inverted onto whole astrocyte surfaces,astrocyte matrix or larninin substrates and left for 2 days. The numberof Schwann cells per unit distance is given as FIG. 5A, with the maximummigration values as FIG. 5B. Schwann cells migrated poorly uponastrocytes but considerably better upon astrocyte matrix or laminin. Alldata are expressed as the mean±S.E.M. of at least three separatedeterminations, each determination consisting of at least 8-12 fragmentsper group. One way analysis of variance (ANOVA) performed upon themaximum migratory distances revealed a statistical difference <0.001between at least one of the groups. A post hoc multiple comparisons test(Tukey test) revealed significant differences (p<0.01) between thegroups marked with (*) and the control astrocytes.

FIGS. 6A and 6B are graphs depicting the migration of Schwann cells onlaminin, and the effects of astroglial soluble factors as determined bythe inverted-fragmented-coverslip migration assay. 1 mm×2 mm glassfragments laden with fluorescently labeled Schwann cells were invertedonto laminin, and left for 2 days in either serum-free (SF) medium orastrocyte conditioned medium (ACM). The number of Schwann cells per unitdistance is given as FIG. 6A, with the maximum migration values as FIG.6B. Schwann cells were found to migrate less well upon laminin in theabsence of serum (c.f. FIG. 1) and considerably better in ACM. All dataare expressed as the mean±S.E.M. of at least three separatedeterminations, each determination consisting of at least 8-12 fragmentsper group. A student's t-test was performed giving p<0.001 (indicated by*).

FIGS. 7A and 7B are graphs, and FIG. 7C is a diagram, depicting theresults of timelapse analysis. FIG. 7A shows the mean migration rates ofsingle Schwann cells plated onto astrocytic, fibroblastic or lamininsubstrates and filmed for a period of 6 hours. The position of the cellbody was recorded (represented diagramatically as FIG. 7C) every 30minutes and the displacement calculated. The number of cells recordedwas 30 upon astrocytes, 25 upon fibroblasts and 30 upon laminin. Schwanncells were found to migrate fastest on laminin, slowest on astrocytesand slightly faster on fibroblasts. One way ANOVA performed on the datarevealed a statistical difference (p<0.001) between at least one of thegroups. A post hoc multiple comparisons test (Tukey test) revealedsignificant differences (p<0.01) between the groups marked with (*) andthe control astrocytes. FIG. 7B shows the duration of interactionbetween Schwann cells and astrocytes and Schwann cells and fibroblasts.The durations of 50 Schwann cell-astrocyte interactions and 45 Schwanncell-fibroblast interactions were recorded. Interactions between Schwanncells and astrocytes were found to be almost 5 times longer than thosewith fibroblasts. A student's t-test was performed and p<0.001 (*) Alldata are expressed as the mean±S.E.M. Scale bar in 5 C is 50 μm.

FIGS. 8A-E are photographs illustrating Schwann cells colliding withsparsely plated astroglia. This series of photographs was obtained fromthe timelapse videomicroscopy apparatus, each picture preceding the nextby a period of 2 hours. In FIG. 8A, a group of two Schwann cellsencounters astroglia (labeled *). The growth cone of one Schwann cellcontacts an astrocyte (arrow indicates first contact). In FIG. 8B, thefirst Schwann cell process continues to explore the astrocyte surfacewhilst the perikarya of the second Schwann cell contacts anotherastrocyte (arrow). The first contact persists beyond the 8 hours ofrecording. The second contact is more short-lived, although astrocyteand Schwann cell remain in close approximation. Scale bar 20 μm.

FIG. 9 is a histogram depicting the adhesion of Schwann cells to varioussubstrates. 20,000 DiI-labelled Schwann cells were plated onto a 13 mmglass coverslip coated with laminin, a complete monolayer of astrocytes,fibroblasts or Schwann cells and then placed onto a shaking (25 rpm)platform for 30 minutes. After washing, the number of cells found stillto be attached were counted. More cells were found to have stuck on theastrocytic surfaces than on fibroblasts or laminin whereas even more hadstuck to Schwann cells. Note that the speed of migration as determinedby the inverted-fragmented-coverslip migration assay is inverselyproportional to the adhesivity of the substrate. All data are normalizedto control and expressed as the mean±S.E.M. of at least three separatedeterminations. One way analysis of variance (ANOVA) was performed andrevealed a statistical difference <0.001 between at least one of thegroups. A post hoc multiple comparisons test (Tukey test) revealedsignificant differences (p<0.01) between the groups marked with (*) andthe control astrocytes.

FIGS. 10A-C are graphs depicting the effect of lowering extracellularcalcium in reducing Schwann cell-astrocyte adhesion and improvingSchwann cell migration on astrocytes. FIG. 10A shows Schwann cell-astrocyte adhesion in reduced calcium solutions. The adhesion assay wasperformed on astrocytes in normal calcium solution (control), in 0.2 mMcalcium (low calcium) solution and in the presence of 1.6 mM EGTA.Adhesion was greatly reduced in both the latter cases. All data arenormalized to control and expressed as the mean±S.E.M. of at least threeseparate determinations. One way analysis of variance (ANOVA) wasperformed and revealed a statistical difference <0.001 between at leastone of the groups. A post hoc multiple comparisons test (Tukey test)revealed significant differences (p<0.01) between the groups marked with(*) and the control astrocytes. FIGS. 10B and C: Schwann cell migrationon astrocytes with reduced extracellular calcium as determined by theinverted coverslip migration assay. The number of cells migrated perunit distance is shown in FIG. 10B with maximum distances represented inFIG. 10C. Schwann cells were found to have migrated further onastrocytes in the presence of reduced calcium than on astrocytes in thepresence of normal calcium levels. A student's t-test revealedsignificant difference between the maximum distances (*p<0.001).

FIGS. 11A-D are graphs depicting the effect of cadherin disruption byrepresentative modulating agents in reducing intercellular adhesion andpromoting Schwann cell migration on astrocytes. FIG. 11A shows Schwanncell-astrocyte adhesion in the presence of representative modulatingagents. The adhesion assay was performed in the presence of modulatingagent or a similar (control) peptide without the HAV sequence. Themodulating agents were found to significantly reduce Schwanncell-astrocyte adhesion compared to either the non-HAV peptide orcontrol. FIG. 11B shows the ability of N-cadherin blocking antibodies(rabbit anti-N-cadherin CAR sequence antibodies; designated as L7) toreduce Schwann cell adhesion to astrocytes and Schwann cells. Neither arabbit polyclonal antibody directed against N-CAM nor the rabbit serumwere found to alter intercellular adhesion. All data in FIGS. 11A and Bare normalized with respect to control and expressed as the mean±S.E.M.of at least three separate determinations. One way analysis of variance(ANOVA) was performed and revealed a statistical difference <0.001between at least one of the groups. A post hoc multiple comparisons test(Tukey test) revealed significant differences (p<0.01) between thegroups marked with (*) and the control astrocytes. FIGS. 11C and 11D:Increased Schwann cell migration on astrocytes in the presence ofcadherin-function blocking antibodies (L7). FIG. 11C represents numberof cells migrated per unit distance, with FIG. 11D representing maximummigration distance. Only the anti-cadherin CAR sequence antibody L7caused a significant difference as determined by post hoc analysis(Tukey test) following one way ANOVA (*p<0.001). The L7 antibodyincreased migration on astroctyes up to three fold compared to control.

FIGS. 12A-D are photographs showing Schwann cell migration as visualizedby the inverted coverslip migration assay. 1 mm×2 mm glass fragmentsladen with fluorescently labeled Schwann cells were inverted ontovarious substrates and left for 2-3 days. FIG. 12A shows a fluorescentphotograph of Schwann cell migration normally observed on controlastroctyes, with little spread from the edge of the inverted fragment.FIG. 12B shows a fluorescent photograph of Schwann cell migration onskin fibroblasts. Note the considerable number of cells leaving the edgeof the inverted fragment. FIGS. 12C and 12D show a phase and fluorescentphotograph, respectively, of Schwann cell migration on astroctyes in thepresence of the anti-cadherin CAR sequence antiserum (L7). Notice theastrocyte monolayer in FIG. 12C is intact. Scale bar for FIGS. 12A and Bare 40 μm; for FIGS. 12C and 12D the scale bar is 100 μm.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides methods for modulatingcadherin-mediated processes, such as cell adhesion. In general, tomodulate cadherin-mediated cell adhesion, a cadherin-expressing cell iscontacted with a cell adhesion modulating agent (also referred to hereinas a “emodulating agent”) either in vivo or in vitro. A modulating agentmay comprise the classical cadherin cell adhesion recognition (CAR)sequence HAV (ie., His-Ala-Val), with or without one or more additionalCAR sequences, as described below. Alternatively, or in addition, amodulating agent may comprise an antibody, or antigen-binding fragmentthereof, that specifically binds to a cadherin CAR sequence. Withincertain aspects, the methods provided herein inhibit cell adhesion. Suchmethods may generally be used, for example, to treat diseases or otherconditions characterized by undesirable cell adhesion or to facilitatedrug delivery to a specific tissue or tumor. Within other aspects, themethods provided herein may be used to enhance cell adhesion (e.g., tosupplement or replace stitches or to facilitate wound healing). Withinstill further aspects, methods are provided for enhancing and/ordirecting neurite outgrowth. Within one such aspect, the presentinvention provides methods for treating a demyelinating disorder, suchas multiple sclerosis.

Certain aspects of the present invention are based on the discovery thatcadherin-mediated cell adhesion is involved in regulating Schwann celladhesion to astrocytes and in limiting Schwann cell migration. Cadherinsare a rapidly expanding family of cell adhesion molecules (CAMs). Theclassical cadherins are integral membrane glycoproteins that generallypromote cell adhesion through homophilic interactions (a cadherin on thesurface of one cell binds to an identical cadherin on the surface ofanother cell), although cadherins also appear to be capable of formingheterotypic complexes with one another under certain circumstances andwith lower affinity. Cadherins have been shown to regulate epithelial,endothelial, neural and cancer cell adhesion, with different cadherinsexpressed on different cell types. N (neural) - cadherin ispredominantly expressed by neural cells, endothelial cells and a varietyof cancer cell types. E (epithelial) - cadherin is predominantlyexpressed by epithelial cells. Other cadherins are P (placental) -cadherin, which is found in human skin and R (retinal) - cadherin. Adetailed discussion of the classical cadherins is provided in Munro etal., 1996, In: Cell Adhesion and Invasion in Cancer Metastasis, P.Brodt, ed., pp.17-34 (RG Landes Company, Austin Tex.).

The structures of the classical cadherins are generally similar. Asillustrated in FIG. 1, cadherins are composed of five extracellulardomains (EC1-EC5), a single hydrophobic domain (TM) that transverses theplasma membrane (PM), and two cytoplasmic domains (CP1 and CP2). Thecalcium binding motifs DXNDN (SEQ ID NO: 1), DXD and LDRE (SEQ ID NO: 2)are interspersed throughout the extracellular domains. The firstextracellular domain (EC1) contains the classical cadherin CAR sequence,HAV (His-Ala-Val), along with flanking sequences on either side of theCAR sequence that appear to play a role in conferring specificity. Thethree-dimensional solution and crystal structures of the EC1 domain havebeen determined (Overduin et al., Science 267:386-389, 1995; Shapiro etal., Nature 374:327-337, 1995). Sequences of the EC1 domain of somenaturally occurring cadherins are shown in FIG. 2 and SEQ ID NOs: 3 to9.

Cell Adhesion Modulating Agents

The term “cell adhesion modulating agent,” as used herein, refers to amolecule comprising at least one cadherin CAR sequence, generally HAV(His-Ala-Val), and/or an antibody (or antigen-binding fragment thereof)that specifically binds a cadherin CAR sequence. Within embodiments inwhich inhibition of cell adhesion is desired, a modulating agent maycontain one HAV sequence or multiple HAV sequences, which may beadjacent to one another (ie., without intervening sequences) or in closeproximity (ie., separated by peptide and/or non-peptide linkers to givea distance between the CAR sequences that ranges from about 0.1 to 400nm). For example, a modulating agent with adjacent HAV sequences maycomprise the peptide HAVHAV (SEQ ID NO: 10). A representative modulatingagent with HAV sequences in close proximity may comprise the sequenceSHAVSHAVSHAVS (SEQ ID NO: 11). One or more antibodies, or fragmentsthereof, may similarly be used within such embodiments, either alone orin combination with one or more CAR sequences.

A modulating agent as described herein may additionally comprise a CARsequence for one or more different adhesion molecules (including, butnot limited to, other CAMs) and/or one or more antibodies or fragmentsthereof that bind to such sequences. Linkers may, but need not, be usedto separate such CAR sequence(s) and/or antibody sequence(s) from theHAV sequence(s) and/or each other. Such modulating agents may generallybe used within methods in which it is desirable to simultaneouslydisrupt cell adhesion mediated by multiple adhesion molecules. As usedherein, an “adhesion molecule” is any molecule that mediates celladhesion via a receptor on the cell's surface. Adhesion moleculesinclude members of the cadherin gene superfamily that are not classicalcadherins (e.g., proteins that do not contain an HAV sequence and/or oneor more of the other characteristics recited above for classicalcadherins), such as desmogleins (Dsg) and desmocollins (Dsc); integrins;members of the immunoglobulin supergene family, such as N-CAM; and otheruncategorized transmembrane proteins, such as occludin) as well asextracellular matrix proteins such as laminin, fibronectin, collagens,vitronectin, entactin and tenascin. Preferred CAR sequences forinclusion within a modulating agent include Arg-Gly-Asp (RGD), which isbound by integrins (see Cardarelli et al., J. Biol. Chem. 267:23159-64,1992); Tyr-Ile-Gly-Ser-Arg (YIGSR; SEQ ID NO: 12), which is bound byα6β1 integrin; KYSFNYDGSE (SEQ ID NO: 13), which is bound by N-CAM; theN-CAM heparan sulfate-binding site IWKHKGRDVILKKDVRF (SEQ ID NO: 14),the putative Dsc CAR sequences YAT, FAT and YAS; the putative Dsg CARsequence RAL; and/or the putative occludin CAR sequenceGVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQ E (SEQ ID NO: 15), orderivatives thereof such as QSSGSLYGSQ (SEQ ID NO: 16) and QYLYHYCWD(SEQ ID NO: 17).

A linker may be any molecule (including peptide and/or non-peptidesequences as well as single amino acids or other molecules), that doesnot contain a CAR sequence and that can be covalently linked to at leasttwo peptide sequences. Using a linker, HAV-containing peptides and otherpeptide or protein sequences may be joined head-to-tail (i.e., thelinker may be covalently attached to the carboxyl or amino group of eachpeptide sequence), head-to-side chain and/or tail-to-side chain.Modulating agents comprising one or more linkers may form linear orbranched structures. Within one embodiment, modulating agents having abranched structure comprise three different CAR sequences, such as RGD,YIGSR and HAV. Within another embodiment, modulating agents having abranched structure comprise RGD, YIGSR (SEQ ID NO: 12), HAV andKYSFNYDOSE (SEQ ID NO: 13). In a third embodiment, modulating agentshaving a branched structure comprise HAV, YAT, FAT, YAS and RAL.Bi-functional modulating agents that comprise an HAV sequence withflanking E-cadherin-specific sequences joined via a linker to an HAVsequence with flanking N-cadherin-specific sequences are also preferredfor certain embodiments. Linkers preferably produce a distance betweenCAR sequences between 0.1 to 10,000 nm, more preferably about 0.1-400nm. A separation distance between recognition sites may generally bedetermined according to the desired function of the modulating agent.For inhibitors of cell adhesion, the linker distance should be small(0.1-400 nm). For enhancers of cell adhesion, the linker distance shouldbe 400-10,000 nm. One linker that can be used for such purposes is(H₂N(CH₂)_(n)CO₂H)_(m), or derivatives thereof, where n ranges from 1 to10 and m ranges from 1 to 4000. For example, if glycine (H₂NCH₂CO₂H) ora multimer thereof is used as a linker, each glycine unit corresponds toa linking distance of 2.45 angstroms, or 0.245 nm, as determined bycalculation of its lowest energy conformation when linked to other aminoacids using molecular modeling techniques. Similarly, aminopropanoicacid corresponds to a linking distance of 3.73 angstroms, aminobutanoicacid to 4.96 angstroms, aminopentanoic acid to 6.30 angstroms and aminohexanoic acid to 6.12 angstroms. Other linkers that may be used will beapparent to those of ordinary skill in the art and include, for example,linkers based on repeat units of 2,3-diaminopropanoic acid, lysineand/or ornithine. 2,3-Diaminopropanoic acid can provide a linkingdistance of either 2.51 or 3.11 angstroms depending on whether theside-chain amino or terminal amino is used in the linkage. Similarly,lysine can provide linking distances of either 2.44 or 6.95 angstromsand ornithine 2.44 or 5.61 angstroms. Peptide and non-peptide linkersmay generally be incorporated into a modulating agent using anyappropriate method known in the art.

Within embodiments in which enhancement of cell adhesion is desired, amodulating agent may contain multiple HAV sequences, or antibodies thatspecifically bind to such sequences, joined by linkers as describedabove. Enhancement of cell adhesion may also be achieved by attachmentof multiple modulating agents to a support material, as discussedfurther below.

The total number of CAR sequences (including HAV, with or without otherCAR sequences derived from one or more adhesion molecules) presentwithin a modulating agent may range from 1 to a large number, such as100, preferably from 1 to 10, and more preferably from 1 to 5. Peptidemodulating agents comprising multiple CAR sequences typically containfrom 3 to about 1000 amino acid residues, preferably from 4 to 50residues. When non-peptide linkers are employed, each CAR sequence ofthe modulating agent is present within a peptide that generally rangesin size from 3 to 50 residues in length, preferably from 3 to 25residues, more preferably from 3 to 16 residues and still morepreferably from 4 to 16 residues. Additional residue(s) that may bepresent on the N-terminal and/or C-terminal side of a CAR sequence maybe derived from sequences that flank the HAV sequence within one or morenaturally occurring cadherins (e.g., N-cadherin, E-cadherin, P-cadherin,R-cadherin or other cadherins containing the HAV sequence) with orwithout amino acid substitutions and/or other modifications. Flankingsequences for endogenous N-, E-, P- and R-cadherin are shown in FIG. 2,and in SEQ ID NOs: 3 to 9. Alternatively, additional residues present onone or both sides of the CAR sequence(s) may be unrelated to anendogenous sequence (e.g, residues that facilitate purification or othermanipulation and/or residues having a targeting or other function).

A modulating agent may contain sequences that flank the HAV sequence onone or both sides that are designed to confer specificity for celladhesion mediated by one or more specific cadherins, resulting in tissueand/or cell-type specificity. Suitable flanking sequences for conferringspecificity include, but are not limited to, endogenous sequencespresent in one or more naturally occurring cadherins. Modulating agentshaving a desired specificity may be identified using the representativescreens provided herein. Within preferred embodiments, the addition ofappropriate endogenous sequences may result in modulating agents thatspecifically disrupt N-cadherin, P-cadherin or E-cadherin mediated celladhesion. For example, the peptide modulating agent LYSHAVSSNG (SEQ IDNO: 18) or LFSHAVSSNG (SEQ ID NO: 19) may be used to disrupt E-cadherinmediated function, the peptide modulating agent LFGHAVSENG (SEQ ID NO:20) may be used to disrupt P-cadherin mediated function, and the peptideLRAHAVDING (SEQ ID NO: 21) may be used to disrupt N-cadherin mediatedfunction.

To facilitate the preparation of modulating agents having a desiredspecificity, nuclear magnetic resonance (NMR) and computationaltechniques may be used to determine the conformation of a peptide thatconfers a known specificity. NMR is widely used for structural analysisof molecules. Cross-peak intensities in nuclear Overhauser enhancement(NOE) spectra, coupling constants and chemical shifts depend on theconformation of a compound. NOE data provide the interproton distancebetween protons through space. This information may be used tofacilitate calculation of the lowest energy conformation for the HAVsequence. Conformation may then be correlated with tissue specificity topermit the identification of peptides that are similarly tissue specificor have enhanced tissue specificity.

Modulating agents may be polypeptides or salts thereof, containing onlyamino acid residues linked by peptide bonds, or may contain non-peptideregions, such as linkers. Peptide regions of a modulating agent maycomprise residues of L-amino acids, D-amino acids, or any combinationthereof Amino acids may be from natural or non-natural sources, providedthat at least one amino group and at least one carboxyl group arepresent in the molecule; α- and β-amino acids are generally preferred.The 20 L-amino acids commonly found in proteins are identified herein bythe conventional three-letter or one-letter abbreviations indicated inTable 1, and the corresponding D-amino acids are designated by a lowercase one letter symbol.

TABLE 1 Amino acid one-letter and three-letter abbreviations A AlaAlanine R Arg Arginine D Asp Aspartic acid N Asn Asparagine C CysCysteine Q Gln Glutamine E Glu Glutamic acid G Gly Glycine H HisHistidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met Methionine FPhe Phenylalanine P Pro Proline S Ser Serine T Thr Threonine W TrpTryptophan Y Tyr Tyrosine V Val Valine

A modulating agent may also contain rare amino acids (such as4-hydroxyproline or hydroxylysine), organic acids or amides and/orderivatives of common amino acids, such as amino acids having theC-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester)or amidated and/or having modifications of the N-terminal amino group(e.g., acetylation or alkoxycarbonylation), with or without any of awide variety of side-chain modifications and/or substitutions (e.g.,methylation, benzylation, t-butylation, tosylation, alkoxycarbonylation,and the like). Preferred derivatives include amino acids having aC-terminal amide group. Residues other than common amino acids that maybe present with a modulating agent include, but are not limited to,2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid,α-aminoadipic acid, m-aminomethylbenzoic acid and α,β-diaminopropionicacid.

Certain preferred modulating agents for use within the present inventioncomprise at least one of the following sequences: LRAHAVDING (SEQ ID NO:21), LRAHAVDVNG (SEQ ID NO: 22), MRAHAVDING (SEQ ID NO: 23),HLGAHAVDINGNQVET (SEQ ID NO: 24), FHLRAHAVDINGNQV (SEQ ID NO: 25),LYSHAVSSNG (SEQ ID NO: 18), LFSHAVSSNG (SEQ ID NO: 19), LFGHAVSENG (SEQID NO: 20), GHAVSE (SEQ ID NO: 26), AHAVSE (SEQ ID NO: 27), AHAVDI (SEQID NO: 28) and/or SHAVSS (SEQ ID NO: 29), wherein each amino acidresidue may, but need not, be modified as described above. Within oneparticularly preferred group, modulating agents comprise an N-terminalacetyl group and/or a C-terminal amide group. Representative modulatingagents comprising a C-terminal amide group include: LRAHAVDING-NH₂ (SEQID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO: 22), MRAHAVDING-NH₂ (SEQ ID NO:23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH₂ (SEQ IDNO: 25), LYSHAVSSNG-NH₂ (SEQ ID NO: 18), LFSHAVSSNG-NH₂ (SEQ ID NO: 19),LFGHAVSENG-NH₂ (SEQ ID NO: 20), GHAVSE-NH₂ (SEQ ID NO: 26), AHAVSE-NH₂(SEQ ID NO: 27), AHAVDI-NH₂ (SEQ ID NO: 28), SHAVSS-NH₂ (SEQ ID NO: 29)and compounds comprising such sequences or derivatives thereof.Representative modulating agents comprising a N-terminal acetyl groupand a C-terminal amide group include: N-Ac-LRAHAVDING-NH₂ (SEQ ID NO:21), N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQ IDNO: 23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), N-Ac-LYSHAVSSNG-NH₂ (SEQ IDNO: 18), N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19), N-Ac-LFGHAVSENG-NH₂ (SEQID NO: 20), N-Ac-GHAVSE-NH₂ (SEQ ID NO: 26), N-Ac-AHAVSE-NH₂ (SEQ ID NO:27), N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28), N-Ac-SHAVSS-NH₂ (SEQ ID NO: 29)and compounds comprising such sequences or derivatives thereof

Within certain other preferred embodiments, as discussed below,relatively small modulating agents that do not contain significantsequences flanking the HAV sequence (e.g., AHAVSE-NH₂; SEQ ID NO: 27)are preferred for modulating N-cadherin and E-cadherin mediated celladhesion. Such modulating agents can be thought of as “master keys” thatfit into peptide binding sites of each of the different classicalcadherins, and are capable of disrupting cell adhesion of neural cells,endothelial cells, epithelial cells and/or certain cancer cells. Suchmodulating agents may generally by used to specifically modulate celladhesion of neural or other cell types by topical administration or bylinking a suitable targeting agent to the peptide, as discussed below.Such peptides may, but need not, contain an N-acetyl group and/or aC-amide group (e.g., N-Ac-AHAVSE-NH₂; SEQ ID NO: 27).

Peptide modulating agents (and peptide portions of modulating agents) asdescribed herein may be synthesized by methods well known in the art,including chemical synthesis and recombinant DNA methods. For modulatingagents up to about 50 residues in length, chemical synthesis may beperformed using solid phase peptide synthesis techniques, in which apeptide linkage occurs through the direct condensation of the α-aminogroup of one amino acid with the α-carboxy group of the other amino acidwith the elimination of a water molecule. Peptide bond synthesis bydirect condensation, as formulated above, requires suppression of thereactive character of the amino group of the first and of the carboxylgroup of the second amino acid. The masking substituents must permittheir ready removal, without inducing breakdown of the labile peptidemolecule.

Solid phase peptide synthesis uses an insoluble polymer for supportduring organic synthesis. The polymer-supported peptide chain permitsthe use of simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc. 85:2149, 1963, which involves assembling a linear peptidechain on a resin support using protected amino acids. Solid phasepeptide synthesis typically utilizes either the Boc or Fmoc strategy.The Boc strategy uses a 1% cross-linked polystyrene resin. The standardprotecting group for α-amino functions is the tert-butyloxycarbonyl(Boc) group. This group can be removed with dilute solutions of strongacids such as 25% trifluoroacetic acid (TFA). The next Boc-amino acid istypically coupled to the amino acyl resin using dicyclohexylcarbodiimide(DCC). Following completion of the assembly, the peptide-resin istreated with anhydrous HF to cleave the benzyl ester link and liberatethe free peptide. Side-chain functional groups are usually blockedduring synthesis by benzyl-derived blocking groups, which are alsocleaved by HF. The free peptide is then extracted from the resin with asuitable solvent, purified and characterized. Newly synthesized peptidescan be purified, for example, by gel filtration, HPLC, partitionchromatography and/or ion-exchange chromatography, and may becharacterized by, for example, mass spectrometry or amino acid sequenceanalysis. In the Boc strategy, C-terminal amidated peptides can beobtained using benzhydrylamine or methylbenzhydrylamine resins, whichyield peptide amides directly upon cleavage with HF.

In the procedures discussed above, the selectivity of the side-chainblocking groups and of the peptide-resin link depends upon thedifferences in the rate of acidolytic cleavage. Orthoganol systems havebeen introduced in which the side-chain blocking groups and thepeptide-resin link are completely stable to the reagent used to removethe α-protecting group at each step of the synthesis. The most common ofthese methods involves the 9-fluorenylmethyloxycarbonyl (Fmoc) approach.Within this method, the side-chain protecting groups and thepeptide-resin link are completely stable to the secondary amines usedfor cleaving the N-α-Fmoc group. The side-chain protection and thepeptide-resin link are cleaved by mild acidolysis. The repeated contactwith base makes the Merrifield resin unsuitable for Fmoc chemistry, andp-alkoxybenzyl esters linked to the resin are generally used.Deprotection and cleavage are generally accomplished using TFA.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the entiresynthesis. In addition, solid phase synthesis is generally most suitablewhen peptides are to be made on a small scale.

N-acetylation of the N-terminal residue can be accomplished by reactingthe final peptide with acetic anhydride before cleavage from the resin.C-amidation may be accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

For longer modulating agents, recombinant methods are preferred forsynthesis. Within such methods, all or part of a modulating agent can besynthesized in living cells, using any of a variety of expressionvectors known to those of ordinary skill in the art to be appropriatefor the particular host cell. Suitable host cells may include bacteria,yeast cells, mammalian cells, insect cells, plant cells, algae and otheranimal cells (e.g., hybridoma, CHO, myeloma). The DNA sequencesexpressed in this manner may encode portions of an endogenous cadherinor other adhesion molecule. Such sequences may be prepared based onknown cDNA or genomic sequences (see Blaschuk et al., J. Mol. Biol211:679-682, 1990), or from sequences isolated by screening anappropriate library with probes designed based on the sequences of knowncadherins. Such screens may generally be performed as described inSambrook et al., Molecular Cloning. A Laboratory Manual, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y., 1989 (and referencescited therein). Polymerase chain reaction (PCR) may also be employed,using oligonucleotide primers in methods well known in the art, toisolate nucleic acid molecules encoding all or a portion of anendogenous adhesion molecule. To generate a nucleic acid moleculeencoding a desired modulating agent, an endogenous cadherin sequence maybe modified using well known techniques. For example, portions encodingone or more CAR sequences may be joined, with or without separation bynucleic acid regions encoding linkers, as discussed above.Alternatively, portions of the desired nucleic acid sequences may besynthesized using well known techniques, and then ligated together toform a sequence encoding the modulating agent.

As noted above, instead of (or in addition to) an HAV sequence, amodulating agent may comprise an antibody, or antigen-binding fragmentthereof, that specifically binds to a cadherin CAR sequence. As usedherein, an antibody, or antigen-binding fragment thereof, is said to“specifically bind” to a cadherin CAR sequence (with or without flankingamino acids) if it reacts at a detectable level (within, for example, anELISA, as described by Newton et al., Develop. Dynamics 197:1-13, 1993)with a peptide containing that sequence, and does not react detectablywith peptides containing a different CAR sequence or a sequence in whichthe order of amino acid residues in the cadherin CAR sequence and/orflanking sequence is altered.

Within certain aspects of the present invention, modulating agentscomprising polyclonal or monoclonal antibodies may be used to enhanceand/or direct neurite outgrowth. Modulating agents comprising antibodiesor antigen-binding fragments thereof (e.g., Fab fragments) may also beused, within other aspects, to inhibit cell adhesion in a variety ofcontexts. For example, such modulating agents may be used for treatmentof demyelinating diseases, such as MS, or to inhibit interactionsbetween tumor cells. Within further aspects, modulating agentscomprising antibodies or antigen-binding fragments thereof that arelinked to one or more linkers, or to a single molecule or supportmaterial may be used to enhance cell adhesion.

Polyclonal and monoclonal antibodies may be raised against a cadherinCAR sequence using conventional techniques. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Inone such technique, an immunogen comprising the cadherin CAR sequence isinitially injected into any of a wide variety of mammals (e.g., mice,rats, rabbits, sheep or goats). The smaller immunogens (ie., less thanabout 20 amino acids) should be joined to a carrier protein, such asbovine serum albumin or keyhole limpet hemocyanin. Following one or moreinjections, the animals are bled periodically. Polyclonal antibodiesspecific for the CAR sequence may then be purified from such antiseraby, for example, affinity chromatography using the modulating agent orantigenic portion thereof coupled to a suitable solid support.

Monoclonal antibodies specific for the cadherin CAR sequence may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, thesemethods involve the preparation of immortal cell lines capable ofproducing antibodies having the desired specificity from spleen cellsobtained from an animal immunized as described above. The spleen cellsare immortalized by, for example, fusion with a myeloma cell fusionpartner, preferably one that is syngeneic with the immunized animal.Single colonies are selected and their culture supernatants tested forbinding activity against the modulating agent or antigenic portionthereof. Hybridomas having high reactivity and specificity arepreferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies, with or without the use of various techniques knownin the art to enhance the yield. Contaminants may be removed from theantibodies by conventional techniques, such as chromatography, gelfiltration, precipitation, and extraction. Antibodies having the desiredactivity may generally be identified using immunofluorescence analysesof tissue sections, cell or other samples where the target cadherin islocalized.

Within preferred embodiments, such monoclonal antibodies are specificfor particular cadherins (e.g., the antibodies bind to E-cadherin, butdo not bind significantly to N-cadherin, or vise versa). Such antibodiesmay be prepared as described above, using an immunogen that comprises(in addition to the HAV sequence) sufficient flanking sequence togenerate the desired specificity (e.g., 6 amino acids on each side isgenerally sufficient). One representative immunogen is the 15-merFHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), linked to KLH (see Newton et al.,Dev. Dynamics 197:1-13, 1993). To evaluate the specificity of aparticular antibody, representative assays as described herein and/orconventional antigen-binding assays may be employed. Such antibodies maygenerally be used for therapeutic, diagnostic and assay purposes, asdescribed herein. For example, such antibodies may be linked to a drugand administered to a mammal to target the drug to a particularcadherin-expressing cell, such as a leukemic cell in the blood.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; see especially page 309) and digested bypapain to yield Fab and Fc fragments. The Fab and Fc fragments may beseparated by affinity chromatography on protein A bead columns (Harlowand Lane, 1988, pages 628-29).

Evaluation of Modulating Agent Activity

As noted above, modulating agents as described herein are capable ofenhancing or inhibiting cadherin-mediated cell adhesion. The ability ofan agent to modulate cell adhesion may generally be evaluated in vitroby assaying the effect on one or more of the following: (1) Schwanncell-astrocyte adhesion, (2) Schwann cell migration on astrocytemonolayers, (3) neurite outgrowth, (4) adhesion between endothelialcells, (5) adhesion between epithelial cells (e.g., normal rat kidneycells and/or human skin) and/or (6) adhesion between cancer cells. Ingeneral, a modulating agent is an inhibitor of cell adhesion if, withinone or more of these representative assays, contact of the test cellswith the modulating agent results in a discernible disruption of celladhesion. Modulating agents that enhance cell adhesion (e.g., agentscomprising multiple HAV sequences and/or linked to a support material)are considered to be modulators of cell adhesion if they are capable ofenhancing neurite outgrowth as described below or are capable ofpromoting cell adhesion, as judged by plating assays to assessepithelial cell adhesion to a modulating agent attached to a supportmaterial, such as tissue culture plastic.

The effect of a modulating agent on Schwann cell adhesion to astrocytesmay generally be evaluated using a cell adhesion assay. Briefly, Schwanncells fluorescently labeled with Di-I may be plated onto an astrocyticsurface (e.g., a glass coverslip coated with a monolayer of astrocytes)and incubated on a shaking platform (e.g., 25 rpm for 30 minutes) in thepresence and absence of modulating agent (e.g., LRAHAVDING (SEQ ID NO:21) at a concentration of 1 mg/mL). Cells may then be washed (e.g., inHanks medium) to remove non-attached cells. The attached cells may thenbe fixed and counted (e.g., using a fluorescent microscope). In general,1 mg/mL of a modulating agent results in an increase or decrease in celladhesion of at least 50%. This assay evaluates the effect of amodulating agent on N-cadherin mediated cell adhesion.

Schwann cell migration may generally be evaluated using amicro-inverted-coverslip assay. In this assay, a dense Schwann cellculture is established on coverslip fragments and Schwann cell migrationaway from the fragment edge is measured. Briefly, Schwann cellsfluorescently labeled with Di-I may be plated on polylysine- andlaminin-coated fragments of a glass coverslip and allowed to bind to thesurface for 16-18 hours. Cells may then be washed (e.g., in Hanksmedium) to remove non-attached cells, and then inverted, with cellsfacing downward onto an astrocyte-coated surface. Cultures are thenincubated further for 2 days in the presence or absence of modulatingagent (e.g., LRAHAVDING (SEQ ID NO: 21) at a concentration of 1 mg/mL)and fixed. The maximum migration distance from the edge of the coverslipfragment may then be measured. At a level of 1 mg/mL, modulating agentresults in an increase or decrease in the maximum migration distance ofat least 50%. This assay evaluates the effect of a modulating agent onN-cadherin mediated cell adhesion.

Within a representative neurite outgrowth assay, neurons may be culturedon a monolayer of cells (e.g., 3T3) that express N-cadherin. Neuronsgrown on such cells (under suitable conditions and for a sufficientperiod of time) extend longer neurites than neurons cultured on cellsthat do not express N-cadherin. For example, neurons may be cultured onmonolayers of 3T3 cells transfected with cDNA encoding N-cadherinessentially as described by Doherty and Walsh, Curr. Op. Neurobiol.4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994; Hall et al.,Cell Adhesion and Commun. 3:441-450, 1996; Doherty and Walsh, Mol. Cell.Neurosci. 8:99-111, 1994; and Safell et al., Neuron 18:231-242, 1997.Briefly, monolayers of control 3T3 fibroblasts and 3T3 fibroblasts thatexpress N-cadherin may be established by overnight culture of 80,000cells in individual wells of an 8-chamber well tissue culture slide.3000 cerebellar neurons isolated from post-natal day 3 mouse brains maybe cultured for 18 hours on the various monolayers in control media(SATO/2% FCS), or media supplemented with various concentrations of themodulating agent or control peptide. The cultures may then be fixed andstained for GAP43 which specifically binds to the neurons and theirneurites. The length of the longest neurite on each GAP43 positiveneuron may be measured by computer assisted morphometry.

A modulating agent that modulates N-cadherin-mediated cell adhesion mayinhibit or enhance such neurite outgrowth. Under the conditionsdescribed above, the presence of 500 μg/mL of a modulating agent thatdisrupts neural cell adhesion should result in a decrease in the meanneurite length by at least 50%, relative to the length in the absence ofmodulating agent or in the presence of a negative control peptide.Alternatively, the presence of 500 μg/mL of a modulating agent thatenhances neural cell adhesion should result in an increase in the meanneurite length by at least 50%.

Within certain cell adhesion assays, the addition of a modulating agentto cells that express a cadherin results in disruption of cell adhesion.A “cadherin-expressing cell,” as used herein, may be any type of cellthat expresses at least one cadherin on the cell surface at a detectablelevel, using standard techniques such as immunocytochemical protocols(e.g., Blaschuk and Farookhi, Dev. Biol. 136:564-567, 1989).Cadherin-expressing cells include endothelial, epithelial and/or cancercells. For example, such cells may be plated under standard conditionsthat, in the absence of modulating agent, permit cell adhesion. In thepresence of modulating agent (e.g., 500 μg/mL), disruption of celladhesion may be determined visually within 24 hours, by observingretraction of the cells from one another.

For use within one such assay, bovine pulmonary artery endothelial cellsmay be harvested by sterile ablation and digestion in 0.1% collagenase(type II; Worthington Enzymes, Freehold, N.J.). Cells may be maintainedin Dulbecco's minimum essential medium supplemented with 10% fetal calfserum and 1% antibiotic-antimycotic at 37° C. in 7% CO₂ in air. Culturesmay be passaged weekly in trypsin-EDTA and seeded onto tissue cultureplastic at 20,000 cells/cm². Endothelial cultures may be used at 1 weekin culture, which is approximately 3 days after culture confluency isestablished. The cells may be seeded onto coverslips and treated (e.g.,for 30 minutes) with modulating agent or a control compound at, forexample, 500 μg/ml and then fixed with 1% paraformaldehyde. As notedabove, disruption of cell adhesion may be determined visually within 24hours, by observing retraction of the cells from one another. This assayevaluates the effect of a modulating agent on N-cadherin mediated celladhesion.

Within another such assay, the effect of a modulating agent on normalrat kidney (NRK) cells may be evaluated. According to a representativeprocedure, NRK cells (ATCC #1571-CRL) may be plated at 10-20,000 cellsper 35 mm tissue culture flasks containing DMEM with 10% FCS andsub-cultured periodically (Laird et al., J. Cell Biol. 131:1193-1203,1995). Cells may be harvested and replated in 35 mm tissue cultureflasks containing 1 mm coverslips and incubated until 50-65% confluent(24-36 hours). At this time, coverslips may be transferred to a 24-wellplate, washed once with fresh DMEM and exposed to modulating agent at aconcentration of, for example, 1 mg/mL for 24 hours. Fresh modulatingagent may then be added, and the cells left for an additional 24 hours.Cells may be fixed with 100% methanol for 10 minutes and then washedthree times with PBS. Coverslips may be blocked for 1 hour in 2% BSA/PBSand incubated for a further 1 hour in the presence of mouseanti-E-cadherin antibody (Transduction Labs, 1:250 dilution). Primaryand secondary antibodies may be diluted in 2% BSA/PBS. Followingincubation in the primary antibody, coverslips may be washed three timesfor 5 minutes each in PBS and incubated for 1 hour with donkeyanti-mouse antibody conjugated to fluorescein (diluted 1:200). Followingfurther washes in PBS (3×5 min) coverslips can be mounted and viewed byconfocal microscopy.

In the absence of modulating agent, NRK cells form characteristictightly adherent monolayers with a cobblestone morphology in which cellsdisplay a polygonal shape. NRK cells that are treated with a modulatingagent that disrupts E-cadherin mediated cell adhesion may assume anon-polygonal and elongated morphology (ie., a fibroblast-like shape)within 48 hours of treatment with 1 mg/mL of modulating agent. Gapsappear in confluent cultures of such cells. In addition, 1 mg/mL of sucha modulating agent reproducibly induces a readily apparent reduction incell surface staining of E-cadherin, as judged by immunofluorescencemicroscopy (Laird et al., J. Cell Biol. 131:1193-1203, 1995), of atleast 75% within 48 hours.

A third cell adhesion assay involves evaluating the effect of amodulating agent on permeability of adherent epithelial and/orendothelial cell layers. For example, the effect of permeability onhuman skin may be evaluated. Such skin may be derived from a naturalsource or may be synthetic. Human abdominal skin for use in such assaysmay generally be obtained from humans at autopsy within 24 hours ofdeath. Briefly, a modulating agent (e.g., 500 μg/ml) and a test marker(e.g., the fluorescent markers Oregon Green™ and Rhodamine Green™Dextran) may be dissolved in a sterile buffer (e.g, phosphate buffer, pH7.2), and the ability of the marker to penetrate through the skin andinto a receptor fluid (e.g., phosphate buffer) may be measured using aFranz Cell apparatus (Franz, Curr. Prob. Dermatol. 7:58-68, 1978; Franz,J. Invest. Dermatol. 64:190-195, 1975). The penetration of the markersthrough the skin may be assessed at, for example, 6, 12, 24, 36, and 48hours after the start of the experiment. In general, a modulating agentthat enhances the permeability of human skin results in a statisticallysignificant increase in the amount of marker in the receptor compartmentafter 6-48 hours in the presence of 500 μg/mL modulating agent. Thisassay evaluates the effect of a modulating agent on E-cadherin mediatedcell adhesion.

Modulating Agent Modification and Formulations

A modulating agent as described herein may, but need not, be linked toone or more additional molecules. In particular, as discussed below, itmay be beneficial for certain applications to link multiple modulatingagents (which may, but need not, be identical) to a support material,such as a single molecule (e.g., keyhole limpet hemocyanin) or a solidsupport, such as a polymeric matrix (which may be formulated as amembrane or microstructure, such as an ultra thin film), a containersurface (e.g., the surface of a tissue culture plate or the interiorsurface of a bioreactor), or a bead or other particle, which may beprepared from a variety of materials including glass, plastic orceramics. For certain applications, biodegradable support materials arepreferred, such as cellulose and derivatives thereof, collagen, spidersilk or any of a variety of polyesters (e.g., those derived from hydroxyacids and/or lactones) or sutures (see U.S. Pat. No. 5,245,012). Withincertain embodiments, modulating agents and molecules comprising otherCAR sequence(s) (e.g., an RGD sequence) may be attached to a supportsuch as a polymeric matrix, preferably in an alternating pattern.

Suitable methods for linking a modulating agent to a support materialwill depend upon the composition of the support and the intended use,and will be readily apparent to those of ordinary skill in the art.Attachment may generally be achieved through noncovalent association,such as adsorption or affinity or, preferably, via covalent attachment(which may be a direct linkage between a modulating agent and functionalgroups on the support, or may be a linkage by way of a cross-linkingagent). Attachment of a modulating agent by adsorption may be achievedby contact, in a suitable buffer, with a solid support for a suitableamount of time. The contact time varies with temperature, but isgenerally between about 5 seconds and 1 day, and typically between about10 seconds and 1 hour.

Covalent attachment of a modulating agent to a molecule or solid supportmay generally be achieved by first reacting the support material with abifunctional reagent that will also react with a functional group, suchas a hydroxyl, thiol, carboxyl, ketone or amino group, on the modulatingagent. For example, a modulating agent may be bound to an appropriatepolymeric support or coating using benzoquinone, by condensation of analdehyde group on the support with an amine and an active hydrogen onthe modulating agent or by condensation of an amino group on the supportwith a carboxylic acid on the modulating agent. A preferred method ofgenerating a linkage is via amino groups using glutaraldehyde. Amodulating agent may be linked to cellulose via ester linkages.Similarly, amide linkages may be suitable for linkage to other moleculessuch as keyhole limpet hemocyanin or other support materials. Multiplemodulating agents and/or molecules comprising other CAR sequences may beattached, for example, by random coupling, in which equimolar amounts ofsuch molecules are mixed with a matrix support and allowed to couple atrandom.

Although modulating agents as described herein may preferentially bindto specific tissues or cells, and thus may be sufficient to target adesired site in vivo, it may be beneficial for certain applications toinclude an additional targeting agent. Accordingly, a targeting agentmay also, or alternatively, be linked to a modulating agent tofacilitate targeting to one or more specific tissues. As used herein, a“targeting agent,” may be any substance (such as a compound or cell)that, when linked to a modulating agent enhances the transport of themodulating agent to a target tissue, thereby increasing the localconcentration of the modulating agent. Targeting agents includeantibodies or fragments thereof, receptors, ligands and other moleculesthat bind to cells of, or in the vicinity of, the target tissue. Knowntargeting agents include serum hormones, antibodies against cell surfaceantigens, lectins, adhesion molecules, tumor cell surface bindingligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes andthose drugs and proteins that bind to a desired target site. Among themany monoclonal antibodies that may serve as targeting agents areanti-TAC, or other interleukin-2 receptor antibodies; 9.2.27 andNR-ML-05, reactive with the 250 kilodalton human melanoma-associatedproteoglycan; and NR-LU-10, reactive with a pancarcinoma glycoprotein.An antibody targeting agent may be an intact (whole) molecule, afragment thereof, or a functional equivalent thereof. Examples ofantibody fragments are F(ab′)2, -Fab′, Fab and F[v] fragments, which maybe produced by conventional methods or by genetic or proteinengineering. Linkage is generally covalent and may be achieved by, forexample, direct condensation or other reactions, or by way of bi- ormulti-functional linkers. Within other embodiments, it may also bepossible to target a polynucleotide encoding a modulating agent to atarget tissue, thereby increasing the local concentration of modulatingagent. Such targeting may be achieved using well known techniques,including retroviral and adenoviral infection.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a modulating agent. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds. Theuse of certain specific drugs within the context of the presentinvention is discussed below.

Modulating agents as described herein may be present within apharmaceutical composition. A pharmaceutical composition comprises oneor more modulating agents in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/orpreservatives. Within yet other embodiments, compositions of the presentinvention may be formulated as a lyophilizate. One or more modulatingagents (alone or in combination with a targeting agent and/or drug) may,but need not, be encapsulated within liposomes using well knowntechnology. Compositions of the present invention may be formulated forany appropriate manner of administration, including for example,topical, oral, nasal, intravenous, intracranial, intraperitoneal,subcutaneous, or intramuscular administration.

For certain embodiments, as discussed below, a pharmaceuticalcomposition may further comprise a modulator of cell adhesion that ismediated by one or more molecules other than cadherins. Such modulatorsmay generally be prepared as described above, incorporating one or morenon-cadherin CAR sequences and/or antibodies thereto in place of thecadherin CAR sequences and antibodies. Such compositions areparticularly useful for situations in which it is desirable to inhibitcell adhesion mediated by multiple cell-adhesion molecules, such asother members of the cadherin gene superfamily that are not classicalcadherins (e.g., Dsg and Dsc); integrins; members of the immunoglobulinsupergene family, such as N-CAM; and other uncategorized transmembraneproteins, such as occludin, as well as extracellular matrix proteinssuch as laminin, fibronectin, collagens, vitronectin, entactin andtenascin. Preferred CAR sequences for use within such a modulatorinclude RGD, YIGSR (SEQ ID NO: 12), KYSFNYDGSE (SEQ ID NO: 13),IWKHKGRDVILKKDVRF (SEQ ID NO: 14), YAT, FAT, YAS, RAL and/orGVNPTAQSSGSLYGSQIYALCNQFYTP AATGLYVDQYLYHYCVVDPQE (SEQ ID NO: 15), orderivatives thereof such as QSSGSLYGSQ (SEQ ID NO: 16) and QYLYHYCVVD(SEQ ID NO: 17).

A pharmaceutical composition may also, or alternatively, contain one ormore drugs, which may be linked to a modulating agent or may be freewithin the composition. Virtually any drug may be administered incombination with a modulating agent as described herein, for a varietyof purposes as described below. Examples of types of drugs that may beadministered with a modulating agent include analgesics, anesthetics,antianginals, antifungals, antibiotics, anticancer drugs (e.g., taxol ormitomycin C), antiinflammatories (e.g., ibuprofen and indomethacin),anthelmintics, antidepressants, antidotes, antiemetics, antihistamines,antihypertensives, antimalarials, antimicrotubule agents (e.g.,colchicine or vinca alkaloids), antimigraine agents, antimicrobials,antiphsychotics, antipyretics, antiseptics, anti-signaling agents (e.g.,protein kinase C inhibitors or inhibitors of intracellular calciummobilization), antiartiritics, antithrombin agents, antituberculotics,antitussives, antivirals, appetite suppressants, cardioactive drugs,chemical dependency drugs, cathartics, chemotherapeutic agents,coronary, cerebral or peripheral vasodilators, contraceptive agents,depressants, diuretics, expectorants, growth factors, hormonal agents,hypnotics, immunosuppression agents, narcotic antagonists,parasympathomimetics, sedatives, stimulants, sympathomimetics, toxins(e.g., cholera toxin), tranquilizers and urinary antiinfectives.

For imaging purposes, any of a variety of diagnostic agents may beincorporated into a pharmaceutical composition, either linked to amodulating agent or free within the composition. Diagnostic agentsinclude any substance administered to illuminate a physiologicalfunction within a patient, while leaving other physiological functionsgenerally unaffected. Diagnostic agents include metals, radioactiveisotopes and radioopaque agents (e.g., gallium, technetium, indium,strontium, iodine, barium, bromine and phosphorus-containing compounds),radiolucent agents, contrast agents, dyes (e.g., fluorescent dyes andchromophores) and enzymes that catalyze a colorimetric or fluorometricreaction. In general, such agents may be attached using a variety oftechniques as described above, and may be present in any orientation.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of modulating agent followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a modulating agentdispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane (see, e.g., European PatentApplication 710,491 A). Carriers for use within such formulations arebiocompatible, and may also be biodegradable; preferably the formulationprovides a relatively constant level of modulating agent release. Theamount of modulating agent contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release and the nature of the condition to be treated orprevented.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented).Appropriate dosages and a suitable duration and frequency ofadministration will be determined by such factors as the condition ofthe patient, the type and severity of the patient's disease and themethod of administration. In general, an appropriate dosage andtreatment regimen provides the modulating agent(s) in an amountsufficient to provide therapeutic and/or prophylactic benefit. Withinparticularly preferred embodiments of the invention, a modulating agentor pharmaceutical composition as described herein may be administered ata dosage ranging from 0.001 to 50 mg/kg body weight, preferably from 0.1to 20 mg/kg, on a regimen of single or multiple daily doses. For topicaladministration, a cream typically comprises an amount of modulatingagent ranging from 0.00001% to 1%, preferably 0.0001% to 0.002%. Fluidcompositions typically contain an amount of modulating agent rangingfrom 10 ng/ml to 5 mg/ml, preferably from 10 μg to 2 mg/mL. Appropriatedosages may generally be determined using experimental models and/orclinical trials. In general, the use of the minimum dosage that issufficient to provide effective therapy is preferred. Patients maygenerally be monitored for therapeutic effectiveness using assayssuitable for the condition being treated or prevented, which will befamiliar to those of ordinary skill in the art.

Modulating Agent Methods of Use

In general, the modulating agents and compositions described herein maybe used for modulating the adhesion of cadherin-expressing cells (i.e.,cells that express one or more of E-cadherin, N-cadherin, P-cadherin,R-cadherin and/or other cadherin(s) containing the HAV sequence,including as yet undiscovered cadherins) in vitro and/or in vivo. Asnoted above, modulating agents for purposes that involve the disruptionof cadherin-mediated cell adhesion may comprise an HAV sequence,multiple HAV sequences in close proximity and/or an antibody (or anantigen-binding fragment thereof) that recognizes a cadherin CARsequence. When it is desirable to also disrupt cell adhesion mediated byother adhesion molecules, a modulating agent may additionally compriseone or more CAR sequences bound by such adhesion molecules (and/orantibodies or fragments thereof that bind such sequences), preferablyseparated from each other and from the HAV sequence by linkers. As notedabove, such linkers may or may not comprise one or more amino acids. Forenhancing cell adhesion, a modulating agent may contain multiple HAVsequences or antibodies (or fragments), preferably separated by linkers,and/or may be linked to a single molecule or to a support material asdescribed above.

Certain methods involving the disruption of cell adhesion as describedherein have an advantage over prior techniques in that they permit thepassage of molecules that are large and/or charged across barriers ofcadherin-expressing cells. As described in greater detail below,modulating agents as described herein may also be used to disrupt orenhance cell adhesion in a variety of other contexts. Within each of themethods described herein, one or more modulating agents may generally beadministered alone, or within a pharmaceutical composition. In eachspecific method described herein, as noted above, a targeting agent maybe employed to increase the local concentration of modulating agent atthe target site.

Within one aspect, one or more modulating agents may be used for therapyof a demyelinating neurological disease in a mammal. There are a numberof demyelinating diseases, such as multiple sclerosis, characterized byoligodendrocyte death. It has been found, within the context of thepresent invention, that Schwann cell migration on astrocytes isinhibited by N-cadherin. Modulating agents that disrupt N-cadherinmediated cell adhesion as described herein, when implanted with Schwanncells into the central nervous system, may facilitate Schwann cellmigration and permit the practice of Schwann cell replacement therapy.

Multiple sclerosis patients suitable for treatment may be identified bycriteria that establish a diagnosis of clinically definite or clinicallyprobable MS (see Poser et al., Ann. Neurol. 13:227, 1983). Candidatepatients for preventive therapy may be identified by the presence ofgenetic factors, such as HLA-type DR2a and DR2b, or by the presence ofearly disease of the relapsing remitting type.

Schwann cell grafts may be implanted directly into the brain along withthe modulating agent(s) using standard techniques. Preferred peptidemodulating agents for use within such methods include LRAHAVDING-NH₂(SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO: 22), MRAHAVDING-NH₂ (SEQ IDNO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH₂ (SEQID NO: 25), AHAVSE-NH₂ (SEQ ID NO: 27), AHAVDI-NH₂ (SEQ ID NO: 28),N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO:22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQID NO: 24), N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), N-Ac-AHAVSE-NH₂(SEQ ID NO: 27), N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28), derivatives of suchsequences and modulating agents comprising any one of these sequences orderivatives thereof. Preferred antibody modulating agents include Fabfragments directed against the N-cadherin CAR sequenceFHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25). Such antibodies and fragments canbe prepared using standard techniques, as discussed above. Suitableamounts of modulating agent generally range as described above,preferably from about 10 μg/mL to about 1 mg/mL.

Alternatively, a modulating agent may be implanted with oligodendrocyteprogenitor cells (OPs) derived from donors not afflicted with thedemyelinating disease. The myelinating cell of the CNS is theoligodendrocyte. Although mature oligodendrocytes and immature cells ofthe oligodendrocyte lineage, such as the oligodendrocyte type 2astrocyte progenitor, have been used for transplantation, OPs are morewidely used. OPs are highly motile and are able to migrate fromtransplant sites to lesioned areas where they differentiate into maturemyelin-forming oligodendrocytes and contribute to repair of demyelinatedaxons (see e.g., Groves et al., Nature 362:453-55, 1993; Baron-VanEvercooren et al., Glia 16:147-64, 1996). OPs can be isolated usingroutine techniques known in the art (see e.g., Milner andFrench-Constant, Development 120:3497-3506, 1994), from many regions ofthe CNS including brain, cerebellum, spinal cord, optic nerve andolfactory bulb. Substantially greater yields of OP's are obtained fromembryonic or neonatal rather than adult tissue. OPs may be isolated fromhuman embryonic spinal cord and cultures of neurospheres established.Human fetal tissue is a potential valuable and renewable source of donorOP's for future, long range transplantation therapies of demyelinatingdiseases such as MS.

OPs can be expanded in vitro if cultured as “homotypic aggregates” or“spheres” (Avellana-Adalid et al, J. Neurosci. Res. 45:558-70, 1996).Spheres (sometimes called “oligospheres” or “neurospheres”) are formedwhen OPs are grown in suspension in the presence of growth factors suchas PDGF and FGF. OPs can be harvested from spheres by mechanicaldissociation and used for subsequent transplantation or establishment ofnew spheres in culture. Alternatively, the spheres themselves may betransplanted, providing a “focal reservoir” of OPs (Avellana-Adalid etal, J. Neurosci. Res. 45:558-70, 1996).

An alternative source of OP may be spheres derived from CNS stem cells.Recently, Reynolds and Weiss, Dev. Biol. 165:1-13, 1996 have describedspheres formed from EGF-responsive cells derived from embryonicneuroepithelium, which appear to retain the pluripotentiality exhibitedby neuroepithelium in vivo. Cells dissociated from these spheres areable to differentiate into neurons, oligodendrocytes and astrocytes whenplated on adhesive substrates in the absence of EGF, suggesting thatEGF-responsive cells derived from undifferentiated embryonicneuroepithelium may represent CNS stem cells (Reynolds and Weiss, Dev.Biol. 165:1-13, 1996). Spheres derived from CNS stem cells provide analternative source of OP which may be manipulated in vitro fortransplantation in vivo. Spheres composed of CNS stem cells may furtherprovide a microenvironment conducive to increased survival, migration,and differentiation of the OPs in vivo.

The use of neurospheres for the treatment of MS may be facilitated bymodulating agents that enhance cell migration from the spheres. In theabsence of modulating agent, the cells within the spheres adhere tightlyto one another and migration out of the spheres is hindered. Modulatingagents that disrupt N-cadherin mediated cell adhesion as describedherein, when injected with neurospheres into the central nervous system,may improve cell migration and increase the efficacy of OP replacementtherapy.

Neurosphere grafts may be implanted directly into the central nervoussystem along with the modulating agent(s) using standard techniques.Preferred peptide modulating agents for use within such methods includeLRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO: 22),MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), AHAVSE-NH₂ (SEQ ID NO: 27),AHAVDI-NH₂ (SEQ ID NO: 28), N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21),N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO:23), N-Ac-HLGAHAVDINONQVET-NH₂ (SEQ ID NO: 24), N-Ac-FHLRAHAVDINGNQV-NH₂(SEQ ID NO: 25), N-Ac-AHAVSE-NH₂ (SEQ ID NO: 27), N-Ac-AHAVDI-NH₂ (SEQID NO: 28) and derivatives of such sequences. Modulating agentscomprising one or more of these sequences or derivatives thereof arealso preferred. Preferred antibody modulating agents include Fabfragments directed against the N-cadherin CAR sequenceFHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25). Such antibodies and fragments canbe prepared using standard techniques, as discussed above. Suitableamounts of modulating agent generally range as described above,preferably from about 10 μg/mL to about 1 mg/mL.

Alternatively, a modulating agent may be administered alone or within apharmaceutical composition. The duration and frequency of administrationwill be determined by such factors as the condition of the patient, andthe type and severity of the patient's disease. Within particularlypreferred embodiments of the invention, the modulating agent orpharmaceutical composition may be administered at a dosage ranging from0.1 mg/kg to 20 mg/kg although appropriate dosages may be determined byclinical trials. Methods of administration include injection,intravenous or intrathecal (ie., directly in cerebrospinal fluid). Amodulating agent or pharmaceutical composition may further comprise adrug (e.g, an immunomodulatory drug).

Effective treatment of multiple sclerosis may be evidenced by any of thefollowing criteria: EDSS (extended disability status scale), appearanceof exacerbations or MRI (magnetic resonance imaging). The EDSS is ameans to grade clinical impairment due to MS (Kurtzke, Neurology33:1444, 1983), and a decrease of one full step defines an effectivetreatment in the context of the present invention (Kurtzke, Ann. Neurol.36:573-79, 1994). Exacerbations are defined as the appearance of a newsymptom that is attributable to MS and accompanied by an appropriate newneurologic abnormality (Sipe et al., Neurology 34:1368, 1984). Therapyis deemed to be effective if there is a statistically significantdifference in the rate or proportion of exacerbation-free patientsbetween the treated group and the placebo group or a statisticallysignificant difference in the time to first exacerbation or duration andseverity in the treated group compared to control group. MRI can be usedto measure active lesions using gadolinium-DTPA-enhanced imaging(McDonald et al. Ann. Neurol. 36:14, 1994) or the location and extent oflesions using T₂-weighted techniques. The presence, location and extentof MS lesions may be determined by radiologists using standardtechniques. Improvement due to therapy is established when there is astatistically significant improvement in an individual patient comparedto baseline or in a treated group versus a placebo group.

Efficacy of the modulating agent in the context of prevention may bejudged based on clinical measurements such as the relapse rate and EDSS.Other criteria include a change in area and volume of T2 images on MRI,and the number and volume of lesions determined by gadolinium enhancedimages.

Within other aspects, methods are provided in which cell adhesion isdiminished. In one such aspect, the present invention provides methodsfor reducing unwanted cellular adhesion by administering a modulatingagent as described herein. Unwanted cellular adhesion can occur betweentumor cells, between tumor cells and normal cells or between normalcells as a result of surgery, injury, chemotherapy, disease,inflammation or other condition jeopardizing cell viability or function.Preferred modulating agents for use within such methods includeLRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO: 22),MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), LYSHAVSSNG-NH₂ (SEQ ID NO: 18),AHAVSE-NH₂ (SEQ ID NO: 27), AHAVDI-NH₂ (SEQ ID NO: 28), SHAVSS-NH₂ (SEQID NO: 29), LFSHAVSSNG-NH₂ (SEQ ID NO: 19), derivatives of suchsequences (e.g., N-Ac-LRlAHAVDING-NH₂ (SEQ ID NO: 21),N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO:23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24), N-Ac-FHLRAHAVDINGNQV-NH₂(SEQ ID NO: 25), N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18),N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19), N-Ac-AHAVSE-NH₂ (SEQ ID NO: 27),N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28), N-Ac-SHAVSS-NH₂ (SEQ ID NO: 29)) andmodulating agents comprising such sequences or derivatives thereof.Preferred antibody modulating agents include Fab fragments directedagainst either the N-cadherin CAR sequence FHLRAHAVDINGNQV-NH₂ (SEQ IDNO: 25) or E-cadherin CAR sequence LFSHAVSSNG-NH₂ (SEQ ID NO: 19). Inaddition, a modulating agent may comprise the sequence RGD, which isbound by integrins, separated from the HAV sequence via a linker.Alternatively, a separate modulator of integrin-mediated cell adhesionmay be administered in conjunction with the modulating agent(s), eitherwithin the same pharmaceutical composition or separately. Topicaladministration of the modulating agent(s) is generally preferred, butother means may also be employed. Preferably, a fluid composition fortopical administration (comprising, for example, physiological saline)comprises an amount of modulating agent as described above, and morepreferably from 10 μg/mL to 1 mg/mL. Creams may generally be formulatedas described above. Topical administration in the surgical field may begiven once at the end of surgery by irrigation of the wound or as anintermittent or continuous irrigation with the use of surgical drains inthe post-operative period or by the use of drains specifically insertedin an area of inflammation, injury or disease in cases where surgerydoes not need to be performed. Alternatively, parenteral ortranscutaneous administration may be used to achieve similar results.

Within another such aspect, methods are provided for enhancing thedelivery of a drug through the skin of a mammal. Transdermal delivery ofdrugs is a convenient and non-invasive method that can be used tomaintain relatively constant blood levels of a drug. In general, tofacilitate drug delivery via the skin, it is necessary to perturbadhesion between the epithelial cells (keratinocytes) and theendothelial cells of the microvasculature. Using currently availabletechniques, only small, uncharged molecules may be delivered across skinin vivo. The methods described herein are not subject to the same degreeof limitation. Accordingly, a wide variety of drugs may be transportedacross the epithelial and endothelial cell layers of skin, for systemicor topical administration. Such drugs may be delivered to melanomas ormay enter the blood stream of the mammal for delivery to other siteswithin the body.

To enhance the delivery of a drug through the skin, a modulating agentas described herein and a drug are contacted with the skin surface.Preferred modulating agents for use within such methods includeLRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO: 22),MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), LYSHAVSSNG-NH₂ (SEQ ID NO: 18),AHAVSE-NH₂ (SEQ ID NO: 27), AHAVDI-NH₂ (SEQ ID NO: 28), SHAVSS-NH₂ (SEQID NO: 29), LFGHAVSENG-NH₂ (SEQ ID NO: 20), LFSHAVSSNG-NH₂ (SEQ ID NO:19), GHAVSE-NH₂ (SEQ ID NO: 26), derivatives of such sequences (e.g.,N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO:22), N-Ac-MRARAVDING-NH₂ (SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQID NO: 24), N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25),N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18), N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO:19), N-Ac-AHAVSE-NH₂ (SEQ ID NO: 27), N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28),N-Ac-SHAVSS-NH₂ (SEQ ID NO: 29)) and modulating agents comprising suchsequences or derivatives thereof Preferred antibody modulating agentsinclude Fab fragments directed against either the N-cadherin CARsequence FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), P-cadherin CAR sequenceLFGHAVSENG-NH₂ (SEQ ID NO: 20) or E-cadherin CAR sequence LFSHAVSSNG-NH₂(SEQ ID NO: 19). Multifunctional modulating agents comprising thecadherin CAR sequence HAV linked to one or more of the Dsc CAR sequencesYAT, FAT and YAS and/or the Dsg CAR sequence RAL may also be used todisrupt epithelial cell adhesion. Alternatively, a separate modulator ofnon-classical cadherin-mediated cell adhesion may be administered inconjunction with the modulating agent(s), either within the samepharmaceutical composition or separately. Contact may be achieved bydirect application of the modulating agent, generally within acomposition formulated as a cream or gel, or using any of a variety ofskin contact devices for transdermal application (such as thosedescribed in European Patent Application No. 566,816 A; U.S. Pat. No.5,613,958; U.S. Pat. No. 5,505,956). A skin patch provides a convenientmethod of administration (particularly for slow-release formulations).Such patches may contain a reservoir of modulating agent and drugseparated from the skin by a membrane through which the drug diff-uses.Within other patch designs, the modulating agent and drug may bedissolved or suspended in a polymer or adhesive matrix that is thenplaced in direct contact with the patient's skin. The modulating agentand drug may then diff-use from the matrix into the skin. Modulatingagent(s) and drug(s) may be contained within the same composition orskin patch, or may be separately administered, although administrationat the same time and site is preferred. In general, the amount ofmodulating agent administered via the skin varies with the nature of thecondition to be treated or prevented, but may vary as described above.Such levels may be achieved by appropriate adjustments to the deviceused, or by applying a cream formulated as described above. Transfer ofthe drug across the skin and to the target tissue may be predicted basedon in vitro studies using, for example, a Franz cell apparatus, andevaluated in vivo by appropriate means that will be apparent to those ofordinary skill in the art. As an example, monitoring of the serum levelof the administered drug over time provides an easy measure of the drugtransfer across the skin.

Transdermal drug delivery as described herein is particularly useful insituations in which a constant rate of drug delivery is desired, toavoid fluctuating blood levels of a drug. For example, morphine is ananalgesic commonly used immediately following surgery. When givenintermittently in a parenteral form (intramuscular, intravenous), thepatient usually feels sleepy during the first hour, is well during thenext 2 hours and is in pain during the last hour because the blood levelgoes up quickly after the injection and goes down below the desirablelevel before the 4 hour interval prescribed for re-injection is reached.Transdermal administration as described herein permits the maintenanceof constant levels for long periods of time (e.g., days), which allowsadequate pain control and mental alertness at the same time. Insulinprovides another such example. Many diabetic patients need to maintain aconstant baseline level of insulin which is different from their needsat the time of meals. The baseline level may be maintained usingtransdermal administration of insulin, as described herein. Antibioticsmay also be administered at a constant rate, maintaining adequatebactericidal blood levels, while avoiding the high levels that are oftenresponsible for the toxicity (e.g., levels of gentamycin that are toohigh typically result in renal toxicity).

Drug delivery by the methods of the present invention also provide amore convenient method of drug administration. For example, it is oftenparticularly difficult to administer parenteral drugs to newborns andinfants because of the difficulty associated with finding veins ofacceptable caliber to catheterize. However, newborns and infants oftenhave a relatively large skin surface as compared to adults. Transdermaldrug delivery permits easier management of such patients and allowscertain types of care that can presently be given only in hospitals tobe given at home. Other patients who typically have similar difficultieswith venous catheterization are patients undergoing chemotherapy orpatients on dialysis. In addition, for patients undergoing prolongedtherapy, transdermal administration as described herein is moreconvenient than parenteral administration.

Transdermal administration as described herein also allows thegastrointestinal tract to be bypassed in situations where parenteraluses would not be practical. For example, there is a growing need formethods suitable for administration of therapeutic small peptides andproteins, which are typically digested within the gastrointestinaltract. The methods described herein permit administration of suchcompounds and allow easy administration over long periods of time.Patients who have problems with absorption through theirgastrointestinal tract because of prolonged ileus or specificgastrointestinal diseases limiting drug absorption may also benefit fromdrugs formulated for transderrnal application as described herein.

Further, there are many clinical situations where it is difficult tomaintain compliance. For example, patients with mental problems (e.g.,patients with Alzheimer's disease or psychosis) are easier to manage ifa constant delivery rate of drug is provided without having to rely ontheir ability to take their medication at specific times of the day.Also patients who simply forget to take their drugs as prescribed areless likely to do so if they merely have to put on a skin patchperiodically (e.g., every 3 days). Patients with diseases that arewithout symptoms, like patients with hypertension, are especially atrisk of forgetting to take their medication as prescribed.

For patients taking multiple drugs, devices for transdermal applicationsuch as skin patches may be formulated with combinations of drugs thatare frequently used together. For example, many heart failure patientsare given digoxin in combination with furosemide. The combination ofboth drugs into a single skin patch facilitates administration, reducesthe risk of errors (taking the correct pills at the appropriate time isoften confusing to older people), reduces the psychological strain oftaking “so many pills,” reduces skipped dosage because of irregularactivities and improves compliance.

The methods described herein are particularly applicable to humans, butalso have a variety of veterinary uses, such as the administration ofgrowth factors or hormones (e.g, for fertility control) to an animal.

As noted above, a wide variety of drugs may be administered according tothe methods provided herein. Some examples of drug categories that maybe administered transdermally include anti-inflammatory drugs (e.g., inarthritis and in other condition) such as all NSAID, indomethacin,prednisone, etc.; analgesics (especially when oral absorption is notpossible, such as after surgery, and when parenteral administration isnot convenient or desirable), including morphine, codeine, Demerol,acetaminophen and combinations of these (e.g., codeine plusacetaminophen); antibiotics such as Vancomycin (which is not absorbed bythe GI tract and is frequently given intravenously) or a combination ofINH and Rifampicin (e.g., for tuberculosis); anticoagulants such asheparin (which is not well absorbed by the GI tract and is generallygiven parenterally, resulting in fluctuation in the blood levels with anincreased risk of bleeding at high levels and risks of inefficacy atlower levels) and Warfarin (which is absorbed by the GI tract but cannotbe administered immediately after abdominal surgery because of thenormal ileus following the procedure); antidepressants (e.g., insituations where compliance is an issue as in Alzheimer's disease orwhen maintaining stable blood levels results in a significant reductionof anti-cholinergic side effects and better tolerance by patients), suchas amitriptylin, imipramin, prozac, etc.; antihypertensive drugs (e.g,to improve compliance and reduce side effects associated withfluctuating blood levels), such as diuretics and beta-blockers (whichcan be administered by the same patch; e.g., furosemide and propanolol);antipsychotics (e.g., to facilitate compliance and make it easier forcare giver and family members to make sure that the drug is received),such as haloperidol and chlorpromazine; and anxiolytics or sedatives(e.g., to avoid the reduction of alertness related to high blood levelsafter oral administration and allow a continual benefit throughout theday by maintaining therapeutic levels constant).

Numerous other drugs may be administered as described herein, includingnaturally occurring and synthetic hormones, growth factors, proteins andpeptides. For example, insulin and human growth hormone, growth factorslike eiythropoietin, interleukins and inteferons may be delivered viathe skin.

Kits for administering a drug via the skin of a mammal are also providedwithin the present invention. Such kits generally comprise a device fortransdermal application (e.g., a skin patch) in combination with, orimpregnated with, one or more modulating agents. A drug may additionallybe included within such kits.

Within a related aspect, the use of modulating agents as describedherein to increase skin permeability may also facilitate sampling of theblood compartment by passive diffusion, permitting detection and/ormeasurement of the levels of specific molecules circulating in theblood. For example, application of one or more modulating agents to theskin, via a skin patch as described herein, permits the patch tofunction like a sponge to accumulate a small quantity of fluidcontaining a representative sample of the serum. The patch is thenremoved after a specified amount of time and analyzed by suitabletechniques for the compound of interest (e.g., a medication, hormone,growth factor, metabolite or marker). Alternatively, a patch may beimpregnated with reagents to permit a color change if a specificsubstance (e.g., an enzyme) is detected. Substances that can be detectedin this manner include, but are not limited to, illegal drugs such ascocaine, HIV enzymes, glucose and PSA. This technology is of particularbenefit for home testing kits.

Within a further aspect, methods are provided for enhancing delivery ofa drug to a tumor in a mammal, comprising administering a modulatingagent in combination with a drug to a tumor-bearing mammal. Modulatingagents for use within such methods include those designed to disruptE-cadherin and/or N-cadherin mediated cell adhesion, such as AHAVDI-NH₂(SEQ ID NO: 28), which is specific for N-cadherin, SHAVSS-NH₂ (SEQ IDNO: 29) and LFSHAVSSNG-NH₂ (SEQ ID NO: 18), which are specific forE-cadherin, AHAVSE-NH₂ (SEQ ID NO: 27) and derivatives thereof. Otherpreferred modulating agents include LRAHAVDING-NH₂ (SEQ ID NO: 21),LRAHAVDVNG-NH₂ (SEQ ID NO: 22), MRAHAVDING-NH₂ (SEQ ID NO: 23),HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH₂ (SEQ ID NO:25), LYSHAVSSNG-NH₂ (SEQ ID NO: 18), derivatives of such sequences(e.g., N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH₂ (SEQ IDNO: 22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH₂(SEQ ID NO: 24), N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25),N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18), N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO:19), N-Ac-AHAVSE-NH₂ (SEQ ID NO: 27), N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28),N-Ac-SHAVSS-NH₂ (SEQ ID NO: 29)) and modulating agents comprising suchsequences or derivatives thereof. Bi-functional modulating agents thatcomprise an HAV sequence with flanking E-cadherin-specific sequencesjoined via a linker to an HAV sequence with flanking N-cadherin-specificsequences are also preferred. Preferably, the peptide portion(s) of amodulating agent comprises 3-16 amino acids, since longer peptides aredifficult to dissolve in aqueous solution and are more likely to bedegraded by peptidases. To achieve specificity for N- or E-cadherinmediated cell adhesion, the peptide portion(s) preferably comprise 4-16amino acids, and more preferably 6-16 amino acids.

In one particularly preferred embodiment, a modulating agent is capableof disrupting cell adhesion mediated by multiple adhesion molecules. Forexample, a single branched modulating agent (or multiple agents linkedto a single molecule or support material) may disrupt E-cadherin,N-cadherin, occludin, Dsc and Dsg mediated cell adhesion, therebydisrupting adherens junctions, tight junctions and desmosomes. Such anagent may comprise the cadherin CAR sequence, HAV, as well as theputative Dsc CAR sequences YAT, FAT, and YAS; the putative Dsg CARsequence RAL; and the putative occludin CAR sequenceGVNPTAQSSGSLYGSQIYALCNQFYTP AATGLYVDQYLYHYCVVDPQE (SEQ ID NO: 15) or aderivative thereof such as QSSGSLYGSQ (SEQ ID NO: 16) or QYLYHYCVVD (SEQID NO: 17). Such agents serve as multifunctional disrupters of celladhesion. Alternatively, a separate modulator of non-classicalcadherin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferred antibody modulating agents includeFab fragments directed against either the N-cadherin CAR sequenceFHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25) or E-cadherin CAR sequenceLFSHAVSSNG-NH₂ (SEQ ID NO: 18). Fab fragments directed against theoccludin CAR sequence GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCWDPQE(SEQ ID NO: 15) may also be employed, either incorporated into amodulating agent or within a separate modulator that is administeredconcurrently.

Preferably, the modulating agent and the drug are formulated within thesame composition or drug delivery device prior to administration. Ingeneral, a modulating agent may enhance drug delivery to any tumor, andthe method of administration may be chosen based on the type of targettumor. For example, injection or topical administration as describedabove may be preferred for melanomas and other accessible tumors (e.g.,metastases from primary ovarian tumors may be treated by flushing theperitoneal cavity with the composition). Other tumors (e.g., bladdertumors) may be treated by injection of the modulating agent and the drug(such as mitomycin C) into the site of the tumor. In other instances,the composition may be administered systemically, and targeted to thetumor using any of a variety of specific targeting agents. Suitabledrugs may be identified by those of ordinary skill in the art based uponthe type of cancer to be treated (e.g., mitomycin C for bladder cancer).In general, the amount of modulating agent administered varies with themethod of administration and the nature of the tumor, within the typicalranges provided above, preferably ranging from about 1 μg/mL to about 2mg/mL, and more preferably from about 1 μg/mL to 1 mg/mL. Transfer ofthe drug to the target tumor may be evaluated by appropriate means thatwill be apparent to those of ordinary skill in the art. Drugs may alsobe labeled (e.g, using radionuclides) to permit direct observation oftransfer to the target tumor using standard imaging techniques.

Within a related aspect, the present invention provides methods fortreating cancer and/or inhibiting metastasis in a mammal. Cancer tumorsare solid masses of cells, growing out of control, which requirenourishment via blood vessels. The formation of new capillaries is aprerequisite for tumor growth and the emergence of metastases.Administration of modulating agents as described herein may disrupt thegrowth of such blood vessels, thereby providing effective therapy forthe cancer and/or inhibiting metastasis. Modulating agents may also beused to treat leukemias. Preferred modulating agents for use within suchmethods include those that disrupt N-cadherin and/or E-cadherin mediatedcell adhesion, such as LRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂(SEQ ID NO: 22), MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂(SEQ ID NO: 24), FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), AHAVSE-NH₂ (SEQ IDNO: 27), AHAVDI-NH₂ (SEQ ID NO: 28), LFSHAVSSNG-NH₂ (SEQ ID NO: 19),SHAVSS-NH₂ (SEQ ID NO: 29), LYSHAVSSNG-NH₂ (SEQ ID NO: 18), derivativesof such sequences (e.g., N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21),N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO:23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24), N-Ac-FHLRAHAVDINONQV-NH₂(SEQ ID NO: 25), N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18),N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19), N-Ac-AHAVSE-NH₂ (SEQ ID NO: 27),N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28), N-Ac-SHAVSS-NH₂ (SEQ ID NO: 29)) andmodulating agents comprising such sequences or derivatives thereof.Preferably, the peptide portion(s) of such modulating agents comprise3-16 amino acids, more preferably 4-16 amino acids, since longerpeptides are difficult to dissolve in aqueous solution and are morelikely to be degraded by peptidases. Preferred antibody modulatingagents include Fab fragments directed against either the N-cadherin CARsequence FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25) or E-cadherin CAR sequenceLFSHAVSSNG-NH₂ (SEQ ID NO: 19). In addition, a modulating agent maycomprise the sequence RGD, which is recognized by integrins, separatedfrom the HAV sequence via a linker. A modulating agent may beadministered alone (e.g., via the skin) or within a pharmaceuticalcomposition. For melanomas and certain other accessible tumors,injection or topical administration as described above may be preferred.For ovarian cancers, flushing the peritoneal cavity with a compositioncomprising one or more modulating agents may prevent metastasis ofovarian tumor cells. Other tumors (e.g., bladder tumors, bronchialtumors or tracheal tumors) may be treated by injection of the modulatingagent into the cavity. In other instances, the composition may beadministered systemically, and targeted to the tumor using any of avariety of specific targeting agents, as described above. In general,the amount of modulating agent administered varies depending upon themethod of administration and the nature of the cancer, but may varywithin the ranges identified above. The effectiveness of the cancertreatment or inhibition of metastasis may be evaluated using well knownclinical observations, such as monitoring the level of serum tumormarkers (e.g, CEA or PSA).

Within a further related aspect, a modulating agent may be used toinhibit angiogenesis (i.e., the growth of blood vessels frompre-existing blood vessels) in a mammal. Inhibition of angiogenesis maybe beneficial, for example, in patients afflicted with diseases such ascancer or arthritis. Preferred modulating agents for inhibition ofangiogenesis include LRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQID NO: 22), MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ IDNO: 24), FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), AHAVDI-NH₂ (SEQ ID NO:28), derivatives of such sequences (e.g., N-Ac-LRAHAVDING-NH₂ (SEQ IDNO: 21), N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQID NO: 23), N-Ac--HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), N-Ac-AHAVDI-NH₂ (SEQ ID NO:28)) and modulating agents comprising such sequences or derivativesthereof Preferred antibody modulating agents include Fab fragmentsdirected against the N-cadherin CAR sequence FHLRAHAVDINGNQV-NH₂ (SEQ IDNO: 25). In addition, a modulating agent for use in inhibitingangiogenesis may comprise the sequence RGD, which is recognized byintegrins, separated from the HAV sequence via a linker. Alternatively,a separate modulator of integrin-mediated cell adhesion may beadministered in conjunction with the modulating agent(s), either withinthe same pharmaceutical composition or separately. The effect of aparticular modulating agent on angiogenesis may generally be determinedby evaluating the effect of the agent on blood vessel formation. Such adetermination may generally be performed, for example, using a chickchorioallantoic membrane assay (Iruela-Arispe et al., Molecular Biologyof the Cell 6:327-343, 1995). Briefly, a modulating agent may beembedded in a mesh composed of vitrogen at one or more concentrations(e.g, ranging from about 5 to 50 μg/mesh). The mesh(es) may then beapplied to chick chorioallantoic membranes. After 24 hours, the effectof the modulating agent may be determined using computer assistedmorphometric analysis. A modulating agent should inhibit angiogenesis byat least 25% at a concentration of 50 μg/mesh.

The addition of a targeting agent as described above may be beneficial,particularly when the administration is systemic. Suitable modes ofadministration and dosages depend upon the condition to be prevented ortreated but, in general, administration by injection is appropriate.Dosages may vary as described above. The effectiveness of the inhibitionmay be evaluated grossly by assessing the inability of the tumors tomaintain their growth and microscopically by observing an absence ofnerves at the periphery of the tumor.

In yet another related aspect, the present invention provides methodsfor inducing apoptosis in a cadherin-expressing cell. In general,patients afflicted with cancer may benefit from such treatment. Certainpreferred modulating agents for use within such methods comprise thesequence LRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO: 22),MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 25),FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), LYSHAVSSNG-NH₂ (SEQ ID NO: 18),AHAVSE-NH₂ (SEQ ID NO: 27), AHAVDI-NH₂ (SEQ ID NO: 28), SHAVSS-NH₂ (SEQID NO: 29), LFSHAVSSNG-NH₂ (SEQ ID NO: 19), derivatives of suchsequences (e.g, N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH₂(SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO: 23),N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24), N-Ac-FILRAHAVDINGNQV-NH₂ (SEQID NO: 25), N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18), N-Ac-AHAVSE-NH₂ (SEQ IDNO: 27), N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28), N-Ac-SHAVSS-NH₂ (SEQ ID NO:29), N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19)) and modulating agentscomprising such sequences or derivatives thereof. In addition, apreferred modulating agent may comprise the an additional CAR sequences,such as the sequence RGD, which is recognized by integrins. As notedabove, such additional sequences may be separated from the HAV sequencevia a linker. Alternatively, a separate modulator of integrin-mediatedcell adhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately. Preferred antibody modulating agents include Fab fragmentsdirected against either the N-cadherin CAR sequence FHLRAHAVDINGNQV-NH₂(SEQ ID NO: 25) or E-cadherin CAR sequence LYSHAVSSNG-NH₂ (SEQ ID NO:18). Administration may be topical, via injection or by other means, andthe addition of a targeting agent may be beneficial, particularly whenthe administration is systemic. Suitable modes of administration anddosages depend upon the location and nature of the cells for whichinduction of apoptosis is desired but, in general, dosages may vary asdescribed above. A biopsy may be performed to evaluate the level ofinduction of apoptosis.

The present invention also provides methods for enhancing drug deliveryto the central nervous system of a mammal. The blood/brain barrier islargely impermeable to most neuroactive agents, and delivery of drugs tothe brain of a mammal often requires invasive procedures. Using amodulating agent as described herein, however, delivery may be by, forexample, systemic administration of a modulating agent-drug-targetingagent combination, injection of a modulating agent (alone or incombination with a drug and/or targeting agent) into the carotid arteryor application of a skin patch comprising a modulating agent to the headof the patient. Certain preferred modulating agents for use within suchmethods are LRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO:22), MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO:24), FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), AHAVSE-NH₂ (SEQ ID NO: 27),AHAVDI-NH₂ (SEQ ID NO: 28), derivatives of such sequences (e.g.,N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO:22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQID NO: 24), N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), N-Ac-AHAVSE-NH₂(SEQ ID NO: 27), N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28)) and modulating agentscomprising such sequences or derivatives thereof. Also preferred arebi-functional modulating agents comprising a cadherin CAR sequence andthe putative occludin CAR sequence GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ ID NO: 15), or derivatives or portions thereof suchas QSSGSLYGSQ (SEQ ID NO: 16) and QYLYHYCVVD (SEQ ID NO: 17), preferablyjoined by a linker. Alternatively, a separate modulator ofoccludin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferably, the peptide portion(s) of suchmodulating agents comprise 3-16 amino acids, more preferably 4-16 aminoacids. Preferred antibody modulating agents include Fab fragmentsdirected against the N-cadherin CAR sequence FHLRAHAVDINGNQV-NH₂ (SEQ IDNO: 25). Fab fragments directed against the occludin CAR sequenceGVNPTAQSSGSLYGSQIYALCNQFYTPAATGLY VDQYLYHYCWDPQE (SEQ ID NO: 15) mayalso be employed, either incorporated into the modulating agent oradministered concurrently as a separate modulator. In general, theamount of modulating agent administered varies with the method ofadministration and the nature of the condition to be treated orprevented, but typically varies as described above. Transfer of the drugto the central nervous system may be evaluated by appropriate means thatwill be apparent to those of ordinary skill in the art, such as magneticresonance imaging (MRI) or PET scan (positron emitted tomography).

The present invention also provides, within further aspects, methods forenhancing and/or directing neurological growth. In one aspect, neuriteoutgrowth may be enhanced and/or directed by contacting a neuron withone or more modulating agents. Preferred modulating agents for usewithin such methods are linked to a polymeric matrix or other supportand/or contain multiple HAV sequences separated by one or more linkers.Peptides that may be linked to a support material (and/or to one anothervia a linker to generate a suitable modulating agent) include, but arenot limited to, LRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ IDNO: 22), MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ IDNO: 24), FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), AHAVDI-NH₂ (SEQ ID NO:28), derivatives of such sequences (e.g., N-Ac-LRAHAVDING-NH₂ (SEQ IDNO: 21), N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), N-Ac-AHAVDI-NH₂ (SEQ ID NO:28)) and modulating agents comprising such sequences or derivativesthereof. In addition, a modulating agent comprising RGD and/or YIGSR(SEQ ID NO: 12), which are bound by integrins, the cadherin CAR sequenceHAV, and/or the N-CAM CAR sequence KYSFNYDGSE (SEQ ID NO: 13) mayfurther facilitate neurite outgrowth. Modulating agents comprisingantibodies, or fragments thereof, may be used within this aspect of thepresent invention without the use of linkers or support materials.Preferred antibody modulating agents include Fab fragments directedagainst the N-cadherin CAR sequence FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25).Fab fragments directed against the N-CAM CAR sequence KYSFNYDGSE (SEQ IDNO: 13) may also be employed, either incorporated into the modulatingagent or administered concurrently as a separate modulator.

The method of achieving contact and the amount of modulating agent usedwill depend upon the location of the neuron and the extent and nature ofthe outgrowth desired. For example, a neuron may be contacted (e.g., viaimplantation) with modulating agent(s) linked to a support material suchas a suture, fiber nerve guide or other prosthetic device such that theneurite outgrowth is directed along the support material. Alternatively,a tubular nerve guide may be employed, in which the lumen of the nerveguide contains a composition comprising the modulating agent(s). Invivo, such nerve guides or other supported modulating agents may beimplanted using well known techniques to, for example, facilitate thegrowth of severed neuronal connections and/or to treat spinal cordinjuries. It will be apparent to those of ordinary skill in the art thatthe structure and composition of the support should be appropriate forthe particular injury being treated. In vitro, a polymeric matrix maysimilarly be used to direct the growth of neurons onto patternedsurfaces as described, for example, in U.S. Pat. No. 5,510,628.

In certain other aspects, the present invention provides methods forenhancing adhesion of cadherin-expressing cells. Within certainembodiments, a modulating agent may be linked to a solid support,resulting in a matrix that comprises multiple modulating agents. Withinone such embodiment, the support is a polymeric matrix to whichmodulating agents and molecules comprising other CAR sequence(s) areattached (e.g., modulating agents and molecules comprising an RGDsequence may be attached to the same matrix, preferably in analternating pattern). Such matrices may be used in contexts in which itis desirable to enhance adhesion mediated by multiple cell adhesionmolecules. Alternatively, the modulating agent itself may comprisemultiple HAV sequences or antibodies (or fragments thereof), separatedby linkers as described above. Either way, the modulating agent(s)function as a “biological glue” to bind multiple cadherin-expressingcells within a variety of contexts.

Within one such aspect, modulating agents comprising multiple HAVsequences and/or multiple modulating agents linked to a single moleculeor support material may be used to enhance wound healing and/or reducescar tissue in a mammal. Peptides that may be linked to a support,and/or to one another via a linker, to generate a suitable modulatingagent include, but are not limited to, LYSHAVSSNG-NH₂ (SEQ ID NO: 18),AHAVSE-NH₂ (SEQ ID NO: 27), SHAVSS-NH₂ (SEQ ID NO: 29), LFSHAVSSNG-NH₂(SEQ ID NO: 19), derivatives of such sequences (e.g.,N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18), N-Ac-AHAVSE-NH₂ (SEQ ID NO: 27),N-Ac-SHAVSS-NH₂ (SEQ ID NO: 29), N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19))and modulating agents comprising such sequences or derivatives thereof.Preferred antibody modulating agents include Fab fragments directedagainst the E-cadherin CAR sequence LFSHAVSSNG-NH₂ (SEQ ID NO: 19).Modulating agents that are linked to a biocompatible and biodegradablematrix such as cellulose or collagen are particularly preferred. For usewithin such methods, a modulating agent should have a free amino orhydroxyl group. The modulating agents are generally administeredtopically to the wound, where they may facilitate closure of the woundand may augment, or even replace, stitches. Similarly, administration ofmatrix-linked modulating agents may facilitate cell adhesion in skingrafting and prosthetic implants, and may prolong the duration andusefulness of collagen injection. In general, the amount ofmatrix-linked modulating agent administered to a wound, graft or implantsite varies with the severity of the wound and/or the nature of thewound, graft, or implant, but may vary as discussed above.Multi-functional modulating agents comprising the cadherin CAR sequence,HAV, the integrin CAR sequence, RGD, as well as the putative Dsc and DsgCAR sequences YAT, FAT, YAS and RAL may also be used as potentstimulators of wound healing and/or to reduce scar tissue.Alternatively, one or more separate modulator of integrin-, Dsc- and/orDsg-mediated cell adhesion may be administered in conjunction with themodulating agent(s), either within the same pharmaceutical compositionor separately.

Within another aspect, one or more modulating agents may be linked tothe interior surface of a tissue culture plate or other cell culturesupport, such as for use in a bioreactor. Such linkage may be performedby any suitable technique, as described above. Modulating agents linkedin this fashion may generally be used to immobilize cadherin-expressingcells. For example, dishes or plates coated with one or more modulatingagents may be used to immobilize cadherin-expressing cells within avariety of assays and screens. Within bioreactors (i.e., systems forlarge scale production of cells or organoids), modulating agents maygenerally be used to improve cell attachment and stabilize cell growth.Modulating agents may also be used within bioreactors to support theformation and function of highly differentiated organoids derived, forexample, from dispersed populations of fetal mammalian cells.Bioreactors containing biomatrices of modulating agent(s) may also beused to facilitate the production of specific proteins.

Modulating agents as described herein may be used within a variety ofbioreactor configurations. In general, a bioreactor is designed with aninterior surface area sufficient to support large numbers of adherentcells. This surface area can be provided using membranes, tubes,microtiter wells, columns, hollow fibers, roller bottles, plates,dishes, beads or a combination thereof. A bioreactor may becompartmentalized. The support material within a bioreactor may be anysuitable material known in the art; preferably, the support materialdoes not dissolve or swell in water. Preferred support materialsinclude, but are not limited to, synthetic polymers such as acrylics,vinyls, polyethylene, polypropylene, polytetrafluoroethylene, nylons,polyurethanes, polyamides, polysulfones and poly(ethyleneterephthalate); ceramics; glass and silica.

Within further aspects, modulating agents as described herein may beused for modulating the immune system of a mammal in any of severalways. Cadherins are expressed on immature B and T cells (thymocytes andbone marrow pre-B cells), as well as on specific subsets of activated Band T lymphocytes and some hematological malignancies (see Lee et al.,J. Immunol. 152:5653-5659, 1994; Munro et al., Cellular Immunol.169:309-312, 1996; Tsutsui et al., J. Biochem. 120:1034-1039, 1996;Cepek et al., Proc. Natl. Acad. Sci. USA 93:6567-6571, 1996). Modulatingagents may generally be used to modulate specific steps within cellularinteractions during an immune response or during the dissemination ofmalignant lymphocytes.

For example, a modulating agent as described herein may be used to treatdiseases associated with excessive generation of otherwise normal Tcells. Without wishing to be bound by any particular theory, it isbelieved that the interaction of cadherins on maturing T cells and Bcell subsets contributes to protection of these cells from programmedcell death. A modulating agent may decrease such interactions, leadingto the induction of programmed cell death. Accordingly, modulatingagents may be used to treat certain types of diabetes and rheumatoidarthritis, particularly in young children where the cadherin expressionon thymic pre-Tcells is greatest.

Modulating agents may also be administered to patients afflicted withcertain skin disorders (such as cutaneous lymphomas), acute B cellleukemia and excessive immune reactions involving the humoral immunesystem and generation of immunoglobulins, such as allergic responses andantibody-mediated graft rejection. In addition, patients withcirculating cadherin-positive malignant cells (e.g., during regimeswhere chemotherapy or radiation therapy is eliminating a major portionof the malignant cells in bone marrow and other lymphoid tissue) maybenefit from treatment with a modulating agent. Such treatment may alsobenefit patients undergoing transplantation with peripheral blood stemcells.

Preferred modulating agents for use within such methods include thosethat disrupt E-cadherin and/or N-cadherin mediated cell adhesion, suchas LRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO: 22),MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), LYSHAVSSNG-NH₂ (SEQ ID NO: 18),AHAVSE-NH₂ (SEQ ID NO: 27), AHAVDI-NH₂ (SEQ ID NO: 28), SHAVSS-NH₂ (SEQID NO: 29), LFSHAVSSNG-NH₂ (SEQ ID NO: 19), derivatives of suchsequences (e.g., N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21),N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO:23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24), N-Ac-FHLRAHAVDINGNQV-NH₂(SEQ ID NO: 25), N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18), N-Ac-AHAVSE-NH₂(SEQ ID NO: 27), N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28), N-Ac-SHAVSS-NH₂ (SEQID NO: 29), N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19)) and modulating agentscomprising such sequences or derivatives thereof. In addition, apreferred modulating agent may comprise one or more additional CARsequences, such as the sequence RGD, which is bound by integrins. Asnoted above, such additional sequence(s) may be separated from the HAVsequence via a linker. Alternatively, a separate modulator ofintegrin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferred antibody modulating agents includeFab fragments directed against either the N-cadherin CAR sequenceFHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25) or E-cadherin CAR sequenceLYSHAVSSNG-NH₂ (SEQ ID NO: 18). Within the above methods, the modulatingagent(s) are preferably administered systemically (usually by injection)or topically. A modulating agent may be linked to a targeting agent. Forexample, targeting to the bone marrow may be beneficial. A suitabledosage is sufficient to effect a statistically significant reduction inthe population of B and/or T cells that express cadherin and/or animprovement in the clinical manifestation of the disease being treated.Typical dosages generally range as described above.

Within further aspects, the present invention provides methods and kitsfor preventing pregnancy in a mammal. In general, disruption ofE-cadherin function prevents the adhesion of trophoblasts and theirsubsequent fusion to form syncitiotrophoblasts. In one embodiment, oneor more modulating agents as described herein may be incorporated intoany of a variety of well known contraceptive devices, such as spongessuitable for intravaginal insertion (see, e.g., U.S. Pat. No. 5,417,224)or capsules for subdermal implantation. Other modes of administrationare possible, however, including transdermal administration, formodulating agents linked to an appropriate targeting agent. Preferredmodulating agents for use within such methods include LYSHAVSSNG-NH₂(SEQ ID NO: 18), AHAVSE-NH₂ (SEQ ID NO: 27), SHAVSS-NH₂ (SEQ ID NO: 29),LFSHAVSSNG-NH₂ (SEQ ID NO: 19), derivatives of such sequences (e.g.,N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18), N-Ac-AHAVSE-NH₂ (SEQ ID NO: 27),N-Ac-SHAVSS-NH₂ (SEQ ID NO: 29) and N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19))and modulating agents comprising such sequences or derivatives thereof.In addition, a preferred modulating agent may comprise additional CARsequences, such as the sequence RGD, which is bound by integrins. Asnoted above, such additional sequences may be separated from the HAVsequence via a linker. Alternatively, a separate modulator ofintegrin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferred antibody modulating agents includeFab fragments directed against the E-cadherin CAR sequenceLFSHAVSSNG-NH₂ (SEQ ID NO: 19). Suitable methods for incorporation intosuch a device depend upon the type of device and are well known in theart. Such devices facilitate administration of the modulating agent(s)to the uterine region and may provide a sustained release of themodulating agent(s). In general, modulating agent(s) may be administeredvia such a contraceptive device at a dosage ranging from 0.1 to 50mg/kg, although appropriate dosages may be determined by monitoring hCGlevels in the urine. hCG is produced by the placenta, and levels of thishormone rise in the urine of pregnant women. The urine hCG levels can beassessed by radio-immunoassay using well known techniques. Kits forpreventing pregnancy generally comprise a contraceptive deviceimpregnated with one or more modulating agents.

Alternatively, a sustained release formulation of one or more modulatingagents may be implanted, typically subdermally, in a mammal for theprevention of pregnancy. Such implantation may be performed using wellknown techniques. Preferably, the implanted formulation provides adosage as described above, although the minimum effective dosage may bedetermined by those of ordinary skill in the art using, for example, anevaluation of hCG levels in the urine of women.

The present invention also provides methods for increasingvasopermeability in a mammal by administering one or more modulatingagents or pharmaceutical compositions. Within blood vessels, endothelialcell adhesion (mediated by N-cadherin) results in decreased vascularpermeability. Accordingly, modulating agents as described herein thatdecrease N-cadherin mediated adhesion may be used to increase vascularpermeability. Particularly preferred modulating agents includeLRAHAVDING-NH₂ (SEQ ID NO: 21), LRAHAVDVNG-NH₂ (SEQ ID NO: 22),MRAHAVDING-NH₂ (SEQ ID NO: 23), HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), AHAVDI-NH₂ (SEQ ID NO: 28),derivatives of such sequences (e.g, N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21),N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO: 22), N-Ac-MRAHAVDING-NH₂ (SEQ ID NO:23), N-Ac-]HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24),N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25), AHAVDI-NH₂ (SEQ ID NO: 28))and modulating agents comprising such sequences or derivatives thereof.Modulating agents comprising antibodies, or fragments thereof, may alsobe used within this aspect of the present invention. Preferred antibodymodulating agents include Fab fragments directed against the N-cadherinCAR sequence FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25).

Within certain embodiments, preferred modulating agents for use withinsuch methods include peptides capable of decreasing both endothelial andtumor cell adhesion. Such modulating agents may be used to facilitatethe penetration of anti-tumor therapeutic or diagnostic agents (e.g.,monoclonal antibodies) through endothelial cell permeability barriersand tumor barriers. For example, a modulating agent may further comprisea sequence such as SHAVSS-NH₂ (SEQ ID NO: 29), LFSHAVSSNG-NH₂ (SEQ IDNO: 19), AHAVSE-NH₂ (SEQ ID NO: 27), LYSHAVSSNG-NH₂ (SEQ ID NO: 18),and/or one or more derivatives of such sequences (e.g., N-Ac-SHAVSS-NH₂(SEQ ID NO: 29), N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19), N-Ac-AHAVSE-NH₂(SEQ ID NO: 27) or N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18)). Bi-functionalmodulating agents that comprise an HAV sequence with flankingE-cadherin-specific sequences joined via a linker to an HAV sequencewith flanking N-cadherin-specific sequences are also preferred.Alternatively, separate modulating agents capable of disrupting N- andE-cadherin mediated adhesion may be administered concurrently.Preferably, the peptide portion(s) of a modulating agent comprises 3-16amino acids, since longer peptides are difficult to dissolve in aqueoussolution and are more likely to be degraded by peptidases.

In one particularly preferred embodiment, a modulating agent is furthercapable of disrupting cell adhesion mediated by multiple adhesionmolecules. Such an agent may comprise the cadherin CAR sequence, HAV, aswell as and RGD sequence and/or the putative occludin CAR sequenceGVNPTAQSSGSLYGSQIYALCNQFYTP AATGLYVDQYLYHYCVVDPQE (SEQ ID NO: 15) or aderivative thereof such as QSSGSLYGSQ (SEQ ID NO: 16) or QYLYHYCWD (SEQID NO: 17). Alternatively, a separate modulator of non-classicalcadherin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferred antibody modulating agents includeFab fragments directed against either the N-cadherin CAR sequenceFHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25) or E-cadherin CAR sequenceLFSHAVSSNG-NH₂ (SEQ ID NO: 18). Fab fragments directed against theoccludin CAR sequence GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVV DPQE(SEQ ID NO: 15) may also be employed, either incorporated into amodulating agent or within a separate modulator that is administeredconcurrently.

Treatment with a modulating agent may be appropriate, for example, priorto administration of an anti-tumor therapeutic or diagnostic agent (e.g.a monoclonal antibody or other macromolecule), an antimicrobial agent oran anti-inflammatory agent, in order to increase the concentration ofsuch agents in the vicinity of the target tumor, organism orinflammation without increasing the overall dose to the patient.Modulating agents for use within such methods may be linked to atargeting agent to further increase the local concentration ofmodulating agent, although systemic administration of a vasoactive agenteven in the absence of a targeting agent increases the perfusion ofcertain tumors relative to other tissues. Suitable targeting agentsinclude antibodies and other molecules that specifically bind to tumorcells or to components of structurally abnormal blood vessels. Forexample, a targeting agent may be an antibody that binds to a fibrindegradation product or a cell enzyme such as a peroxidase that isreleased by granulocytes or other cells in necrotic or inflamed tissues.

Administration via intravenous injection or transdermal administrationis generally preferred. Effective dosages are generally sufficient toincrease localization of a subsequently administered diagnostic ortherapeutic agent to an extent that improves the clinical efficacy oftherapy of accuracy of diagnosis to a statistically significant degree.Comparison may be made between treated and untreated tumor host animalsto whom equivalent doses of the diagnostic or therapeutic agent areadministered. In general, dosages range as described above.

Within a further aspect, modulating agents as described herein may beused for controlled inhibition of synaptic stability, resulting inincreased synaptic plasticity. Within this aspect, administration of oneor more modulating agents may be advantageous for repair processeswithin the brain, as well as learning and memory, in which neuralplasticity is a key early event in the remodeling of synapses. Celladhesion molecules, particularly N-cadherin and E-cadherin, can functionto stabilize synapses, and loss of this function is thought to be theinitial step in the remodeling of the synapse that is associated withlearning and memory (Doherty et al., J. Neurobiology, 26:437-446, 1995;Martin and Kandel, Neuron, 17:567-570, 1996; Fannon and Colman, Neuron,1 7:423-434, 1996). Inhibition of cadherin function by administration ofone or more modulating agents that inhibit cadherin fimction maystimulate learning and memory. Preferred modulating agents for usewithin such methods include those that disrupt E-cadherin and/orN-cadherin mediated cell adhesion, such as LRAHAVDING-NH₂ (SEQ ID NO:21), LRAHAVDVNO-NH₂ (SEQ ID NO: 22), MRAHAVDING-NH₂ (SEQ ID NO: 23),HLGAHAVDINGNQVET-NH₂ (SEQ ID NO: 24), FHLRAHAVDINGNQV-NH₂ (SEQ ID NO:25), LYSHAVSSNG-NH₂ (SEQ ID NO: 18), AHAVSE-NH₂ (SEQ ID NO: 27),AHAVDI-NH₂ (SEQ ID NO: 28), SHAVSS-NH₂ (SEQ ID NO: 29), LFSHAVSSNG-NH₂(SEQ ID NO: 19), derivatives of such sequences (e.g.,N-Ac-LRAHAVDING-NH₂ (SEQ ID NO: 21), N-Ac-LRAHAVDVNG-NH₂ (SEQ ID NO:22), N-Ac-MRA AVDING-NH₂ (SEQ ID NO: 23), N-Ac-HLGAHAVDINGNQVET-NH₂ (SEQID NO: 24), N-Ac-FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25),N-Ac-LYSHAVSSNG-NH₂ (SEQ ID NO: 18), N-Ac-AHAVSE-NH₂ (SEQ ID NO: 27),N-Ac-AHAVDI-NH₂ (SEQ ID NO: 28), N-Ac-S HAVSS-NH₂ (SEQ ID NO: 29), N-Ac-LFSHAVSSNG-NH₂ (SEQ ID NO: 19)) and modulating agents comprising suchsequences or derivatives thereof. In addition, a preferred modulatingagent may comprise one or more additional CAR sequences, such as thesequence RGD, which is bound by integrins and/or the N-CAM CAR sequenceKYSFNYDGSE (SEQ ID NO: 12). As noted above, such additional sequence(s)may be separated from the HAV sequence via a linker. Alternatively, aseparate modulator of integrin and/or N-CAM mediated cell adhesion maybe administered in conjunction with the modulating agent(s), eitherwithin the same pharmaceutical composition or separately. Preferredantibody modulating agents include Fab fragments directed against eitherthe N-cadherin CAR sequence FHLRAHAVDINGNQV-NH₂ (SEQ ID NO: 25) orE-cadherin CAR sequence LFSHAVSSNO-NH₂ (SEQ ID NO: 19). For suchaspects, administration may be via encapsulation into a delivery vehiclesuch as a liposome, using standard techniques, and injection into, forexample, the carotid artery. Alternatively, a modulating agent may belinked to a disrupter of the blood-brain barrier. In general dosagesrange as described above.

Other aspects of the present invention provide methods that employantibodies raised against the modulating agents for diagnostic and assaypurposes. Assays typically involve using an antibody to detect thepresence or absence of a cadherin (free or on the surface of a cell), orproteolytic fragment containing the EC1 domain in a suitable biologicalsample, such as tumor or normal tissue biopsies, blood, lymph node,serum or urine samples, or other tissue, homogenate, or extract thereofobtained from a patient.

There are a variety of assay formats known to those of ordinary skill inthe art for using an antibody to detect a target molecule in a sample.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. For example, the assay may be performed in aWestern blot format, wherein a protein preparation from the biologicalsample is submitted to gel electrophoresis, transferred to a suitablemembrane and allowed to react with the antibody. The presence of theantibody on the membrane may then be detected using a suitable detectionreagent, as described below.

In another embodiment, the assay involves the use of antibodyimmobilized on a solid support to bind to the target cadherin, or aproteolytic fragment containing the EC1 domain and encompassing the CARsequence, and remove it from the remainder of the sample. The boundcadherin may then be detected using a second antibody or reagent thatcontains a reporter group. Alternatively, a competitive assay may beutilized, in which a cadherin is labeled with a reporter group andallowed to bind to the immobilized antibody after incubation of theantibody with the sample. The extent to which components of the sampleinhibit the binding of the labeled cadherin to the antibody isindicative of the reactivity of the sample with the immobilizedantibody, and as a result, indicative of the level of the cadherin inthe sample.

The solid support may be any material known to those of ordinary skillin the art to which the antibody may be attached, such as a test well ina microtiter plate, a nitrocellulose filter or another suitablemembrane. Alternatively, the support may be a bead or disc, such asglass, fiberglass, latex or a plastic such as polystyrene orpolyvinylchloride. The antibody may be immobilized on the solid supportusing a variety of techniques known to those in the art, which are amplydescribed in the patent and scientific literature.

In certain embodiments, the assay for detection of a cadherin in asample is a two-antibody sandwich assay. This assay may be performed byfirst contacting an antibody that has been immobilized on a solidsupport, commonly the well of a microtiter plate, with the biologicalsample, such that the cadherin within the sample is allowed to bind tothe immobilized antibody (a 30 minute incubation time at roomtemperature is generally sufficient). Unbound sample is then removedfrom the immobilized cadherin-antibody complexes and a second antibody(containing a reporter group such as an enzyme, dye, radionuclide,luminescent group, fluorescent group or biotin) capable of binding to adifferent site on the cadherin is added. The amount of second antibodythat remains bound to the solid support is then determined using amethod appropriate for the specific reporter group. The method employedfor detecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.Standards and standard additions may be used to determine the level ofcadherin in a sample, using well known techniques.

The present invention also provides kits for use in such immunoassays.Such kits generally comprise one or more antibodies, as described above.In addition, one or more additional compartments or containers of a kitgenerally enclose elements, such as reagents, buffers and/or washsolutions, to be used in the immunoassay.

Within further aspects, modulating agents or antibodies (or fragmentsthereof) may be used to facilitate cell identification and sorting invitro or imaging in vivo, permitting the selection of cells expressingdifferent cadherins (or different cadherin levels). Preferably, themodulating agent(s) or antibodies for use in such methods are linked toa detectable marker. Suitable markers are well known in the art andinclude radionuclides, luminescent groups, fluorescent groups, enzymes,dyes, constant immunoglobulin domains and biotin. Within one preferredembodiment, a modulating agent linked to a fluorescent marker, such asfluorescein, is contacted with the cells, which are then analyzed byfluorescence activated cell sorting (FACS).

Antibodies or fragments thereof may also be used within screens ofcombinatorial or other nonpeptide-based libraries to identify othercompounds capable of modulating cadherin-mediated cell adhesion. Suchscreens may generally be performed using an ELISA or other method wellknown to those of ordinary skill in the art that detect compounds with ashape and structure similar to that of the modulating agent. In general,such screens may involve contacting an expression library producing testcompounds with an antibody, and detecting the level of antibody bound tothe candidate compounds. Compounds for which the antibody has a higheraffinity may be further characterized as described herein, to evaluatethe ability to modulate cadherin-mediated cell adhesion.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Preparation of Representative Modulating Agents

This Example illustrates the solid phase synthesis of representativepeptide modulating agents.

The peptides were assembled on either methylbenzhydrylamine (MBHA) resin(for the C-terminal amide peptides) or the traditional Merrifield resins(for any C-terminal acid peptides). Bags of a polypropylene meshmaterial were filled with the resin and soaked in dichloromethane. Theresin packets were washed three times with 5% diisopropylethylamine indichloromethane and then washed with dichloromethane. The packets arethen sorted and placed into a Nalgene bottle containing a solution ofthe amino acid of interest in dichloromethane. An equal amount ofdiisopropylcarbodiimide (DIC) in dichloromethane was added to activatethe coupling reaction. The bottle was shaken for one hour to ensurecompletion of the reaction. The reaction mixture was discarded and thepackets washed with DMF. The N-α-Boc was removed by acidolysis using a55% TFA in dichloromethane for 30 minutes leaving the TFA salt of theα-amino group. The bags were washed and the synthesis completed byrepeating the same procedure while substituting for the correspondingamino acid at the coupling step. Acetylation of the N-terminal, wheredesired, was performed by reacting the peptide resins with a solution ofacetic anhydride in dichloromethane in the presence ofdiisopropylethylamine. The peptide was then side-chain deprotected andcleaved from the resin at 0° C. with liquid HF in the presence ofanisole as a carbocation scavenger.

The crude peptides were purified by reversed-phase high-performanceliquid chromatography and characterized by analytical HPLC and by massspectral analysis.

EXAMPLE 2 Establishment of a Model System for Schwann Cell-AstrocyteInteractions

This Example illustrates a cell boundary assay for use in evaluatinginteractions between Schwann cells and astrocytes.

A. Cell Culture

All cells were cultured in Dulbecco's modified Eagle's medium (DMEM;Gibco, Grand Island, NY) supplemented with penicillin/streptomycin (100U/ml; Gibco) and either 10% fetal calf serum (FCS) or serum-free (SF)medium; a modification of Bottenstein's and Sato's (Proc. Natl. Acad.Sci USA 76:514-517, 1979) defined medium with supplements of insulin (5μg/ml; Sigma, St. Louis, Mo.), transferrin (100 μg/ml; Sigma), glutamine(1 mM; ICN/Flow), progesterone (60 ng/ml; Sigma), putrescine (16 μg/ml;Sigma), selenium (160 ng/ml; Sigma), T4 (500 ng/ml; Sigma), T3 (10ng/ml; Sigma), BSA (0.035%; Sigma) and dexamethasone (38 ng/ml; Sigma).

Schwann cells were cultured from neonatal day 2 (P2) sciatic nerve, avariation of the procedure described by Brockes et al., Brain Res.165:105-118, 1979. Nerves were removed and placed in L-15 medium,cleaned of any blood vessels, musculature and their epineurial sheathsand placed into a 34 mm diameter plastic dish containing Trypsin (0.1%;Sigma) and collagenase (0.03%; Sigma). The nerves were cut very finelyusing dissection scissors and placed in an incubator at 37° C. and 10%CO₂ for 30 minutes. Following this incubation an equal volume oftriturating solution (300 mg BSA; Sigma, 1 mg DNAse; Sigma, 50 mgTrypsin inhibitor; Sigma per 100 ml HBSS) was added and the wholemixture gently triturated using a flamed glass pasteur. Having spun downthe cells into a pellet by centrifugation at 1000 rpm for 3-5 minutes,the cells were then resuspended in DMEM with 10% FCS and plated onpoly-lysine (0.01% Sigma) at a density of 5000 cells/mm². On thefollowing day cells were treated with Cytosine arabinoside (Ara-C1×10⁻⁵M; Sigma) for three days. Following a period of two days in normaluntreated FCS the ARA-C was again applied for a further three days. Thefew remaining fibroblast contaminants were then killed via complementmediated lysis using rabbit serum (a gift from R. Oldroyd) and the IgMclass anti-Thy1.1 (1:1000 Serotec, Kidlington, Oxford, UK).Subsequently, the Schwann cells (>98% pure) were maintained in FCSsupplemented with bovine pituitary extract (BPE; 10 μg/ml; Sigma) andforskolin (2 μM, Sigma). These cells were maintained for experimentsuntil two weeks after the treatment with complement.

Primary astrocyte cultures were obtained from neonatal rats (P2) asdescribed by McCarthy and de Vellis, J. Cell. Biol. 85:890-902, 1980.The brains were removed, de-membraned, chopped and then incubated with0.1% trypsin for 30 minutes. The mixture was then triturated intriturating solution and the cells were centrifuged down into a pellet.Having resuspended the cells in FCS they were plated onto poly-lysinecoated plastic at a density of two brains per 75 cc Falcon flask. After6-10 days, the majority of cells of the oligodendrocyte lineage wereremoved by shaking the culture overnight. Skin fibroblasts were obtainedfrom a flap of skin removed from P2 rat neonates. The tissue was choppedusing a sterile blade and then enzymatically dissociated with trypsinand collagenase for 45 minutes. After trituration, the cells wereresuspended in DMEM containing 10% FCS.

Meningeal cell cultures were obtained from the meningeal cell layerwhich was dissected from P2 brains, then treated as described for theastrocytic cultures.

A7 cells, an astrocyte cell line derived from postnatal brain and shownto support axon growth more readily than primary astrocytes (Fok-Seanget al., Brain Res. 698:207-223, 1995), were grown in DMEM containing 10%FCS.

B. Immunofluorescent Staining

Schwann cells were identified by indirect immunofluorescent labelingusing polyclonal anti-growth associated protein 43 (GAP-43; a generousgift from G. Wilkie) and astrocytes were identified by the mousemonoclonal anti-glial fibrillary acidic protein (GFAP; Boelringer,Laval, Quebec). The tissue was fixed in 4% paraformaldehyde for 30minutes, blocked with PBS-Triton X-100 (0.2%) and 5% goat serum and thengiven one hour of incubation with the primary antibody.Rhodamine-conjugated anti-rabbit antibodies (Jackson lmmunoresearchLabs, Inc. Westgrove, Pa.; 1:200) and fluorescein conjugated anti-mouse(1:200) allowed visualization. Fibroblasts were identified with themouse monoclonal anti-Thy1.1 (Serotec, Kidlington, Oxford, UK; 1:1000)using the same staining technique.

C. The Generation of Schwann Cell and Astrocyte Cellular Boundaries

A cell boundary assay was used to study the behavior of two cellpopulations which have the ability to divide and migrate freely, meetinghead on as continuous cellular frontiers. Schwann cells were prepared asa dense cell suspension consisting of 2×10⁶ cells per ml of solution. 70μl of this suspension was placed as a drop at one end of a 2 mmpolylysine coated coverslip. A glass 10 mm×5 mm fragment was taken witha pair of forceps and the drop was smeared towards the center of thecoverslip so as to generate a straight edge to the drop. An equal numberof a different type of cells suspended in an equal volume as the firstdrop was then placed at the opposite end of the coverslip. Using adifferent glass fragment (of similar dimensions) this second drop wasagain smeared towards the center of the coverslip so that the straightedged boundary of this new drop was as close as possible and parallel tothe edge of the first drop without the two drops mixing. The cells wereallowed to attach for 2-3 hours before washing three times in Hanks toremove any non-attached cells. These cultures were then grown for threedays in medium supplemented with serum, BPE and forskolin to provide amaximal mitotic stimulus to the Schwann cells. The cultures were thenfixed in 4% paraforrnaldehyde for 20 minutes prior toimmunohistochemistry. In this way, interactions between populations ofSchwann cells and astroglia, and between populations of Schwann cellsand fibroblasts were studied with respect to the morphology of theircellular territories.

Once confluent cultures of two cell types were established(approximately 200 μm away from one another), the cultures expanded andinteracted with one another along a straight front. The interactionsbetween the two opposing cell types were then analyzed over the courseof several days. The establishment of territories between Schwann cellsand astrocytic cells, and between Schwann cells and fibroblasts wasstudied. In each case, the two populations of cells generally came intocontact after two days. Cultures consisting of Schwann cells andastrocytes were taken for immunohistochemical analysis (n=18). In allcultures, it was evident that Schwann cell and astroglial territoriesremained largely exclusive. The Schwann cells at the boundary were seenin two orientations. In some areas the long axes of the Schwann cellswere parallel to the astrocytic boundary. Here the territories occupiedby the two cell types were completely exclusive. In other areas theboundary was more complex. Groups of Schwann cells had their long axesat right angles to the cell interface, making finger-like projections,and there was often a slight degree of overlap between the twoterritories (FIG. 1D). Time lapse observations indicated that theastrocytes were constantly advancing, sending processes under theSchwann cells, which would then retreat as a group (data not shown).Apart from the distinctive territorial arrangements, it was observedthat astrocytes in contact with Schwann cells displayed a more intensestaining with GFAP and showed hypertrophy of the perikarya, as reportedby previous authors both in vivo and in vitro (Brook et al., Glia9:292-304, 1993; Ghimikar and Eng, Glia 11:367-377, 1994). Schwann cellsand astrocytes cultured by this method were therefore able to establisha structure similar to the peripheral nerve entry zones seen in vivo.

To determine whether the development of these distinctive patterns werea common feature of the manipulations peculiar to this technique, orunique for the cell types, the assay was repeated using Schwann cellsand fibroblasts, cells normally associated with Schwann cell migrationin damaged peripheral nerve. None of the Schwann cell-fibroblastcultures (n=15) displayed the clear territorial exclusion seen inSchwann cell-astrocyte cultures. Similarly, none of the culturesdisplayed the parallel Schwann cell alignment at the boundary or thefinger-like projections. Indeed, Schwann cells were seen to clustertogether rather irregularly and to overlie the fibroblasts. Phasecontrast photographs showing the parallel alignment commonly seen in theSchwann cell-astrocyte co-cultures and the irregular clustering of theSchwann cells upon the fibroblasts are presented in FIGS. 3A and 3B.

D. Migration of Schwann cells on laminin and monolayers

In order to assess the rates of Schwann cell migration on differentsurfaces, the micro-inverted-coverslip migration assay was employed.This is a variation of the technique first described by Fok-Seang etal., Dev. Biol. 171:1-15, 1995. Schwann cells fluorescently labeled withDi-I (25 μg/ml) were evenly plated onto polylysine and laminin coatedfragments of glass coverslip (1×2 mm). After 16-18 hours, the pieces ofglass coated with Di-I labeled Schwann cells were dipped into Hanksthree times to remove any loosely attached cells and then inverted withcells facing downwards onto laminin-coated tissue culture surfaces oronto cell monolayers. These cultures were then incubated for a furthertwo days and fixed for 20 minutes with 4% paraformaldehyde. The maximummigration distance was measured, and the number of cells in bands of 0.1mm progressing outwards from the edge of the coverslip were counted.

In this assay, a dense Schwann cell culture is established on coverslipfragments and their migration away from its edge measured. The assaytherefore measures the ability of Schwann cells to migrate on a surface,and their ability to migrate away from a confluent Schwann cellmonolayer. The migration front of the foremost cells was measured, andthe number of cells against distance of migration plotted. Schwanncell-laden fragments were placed on larinin to give a baseline migrationrate on a favorable defined surface, and larninin controls were done forcomparison on each assay. The average distance of migration on lamininwas 1.02 mm±0.06 (mean±S.E.M.) over three days. Migration assays weredone on four different cell monolayers: (1) astrocytes cultured frompostnatal brain, (2) A7 astrocyte cell line, (3) fibroblasts, and (4)meningeal cells. These results are presented in FIG. 4A.

The mean maximum distance covered by the Schwann cells on an astroglialmonolayer over 3 days was found to be 0.33 mm±0.02. Migration onfibroblasts was 0.99 mm±0.04 (FIG. 4B). Schwann cells can thereforemigrate on fibroblasts almost as rapidly as on laminin, while migrationon astrocytes is much more limited. FIGS. 12A and 12B compare themigration of Schwann cells upon astrocyte surfaces to that uponfibroblast surfaces, showing the migration of fluorescently labeledcells from the edge of the fragment.

Primary cortical astrocyte cultures purified in the manner describedhave been shown to yield type-I astrocyte purities greater than 95%.Contaminating cell types may include microglial cells, meningeal cellsor cells of the oligodendrocyte-lineage. In order to be certain that therestricted migration of Schwann cells on astrocyte cultures was not dueto the presence of small numbers of meningeal cells, which inhibitoligo-precursor migration (Fok-Seang et al., Dev. Biol 171:1-15, 1995),purified meningeal cell cultures extracted from neonatal brain were usedas a migratory substrate. The average distance of migration by Schwanncells on a meningeal cell monolayer was found to be 0.90 mm±0.04. Thisis a degree of Schwann cell migration similar to that achieved onfibroblasts and laminin, and much greater than on astrocytes.

In order to determine whether non-astrocytic contaminants wereresponsible for the non-permissive behavior, the astrocyte cell line A7was used as a migratory substrate. A homogenous astroglial populationpermitted only 0.40 mm±0.03 of Schwann cell migration over the two dayperiod, very similar to that seen on primary astrocyte cultures.

Thus, when confluent cultures of Schwann cells and astrocytes wereplaced so as to confront one another a clear division of territoryresulted, comparable to the peripheral nerve entry zones. Severalmechanisms could be responsible for the segregation of two differentcell types and their failure to migrate over or through one another. Thesimplest is an inhibitory interaction, as is seen when axon growth conesmeet oligodendrocytes, when axons from CNS and PNS meet, or whenoligodendrocyte precursors meet meningeal cells. However, in suchinstances, the exploratory cell process undergoes a sudden andcatastrophic collapse within a few minutes of cell contact, leading towithdrawal of the migrating cell. This “growth cone collapse” did notoccur when Schwann cells met either astrocytes or fibroblasts. A secondreason for failure of cells to mix could be a lack of complementaryadhesion molecules; however Schwann cells adhere more strongly toastrocytes than to fibroblasts or laminin, both of which supportmigration.

The data presented herein demonstrate that Schwann cells form prolongedand firm contacts with astrocytes. Schwann cells are unable to moveuntil these contacts are broken. This behavior is very similar to thatseen when oligodendrocyte precursors encounter astrocytes or when aSchwann cell process encounters an axonal growth cone in the presence ofexternal calcium. Meetings with fibroblasts result in much shorter livedcontacts. The results suggest that a secreted or cell-associated factormay be involved in this interaction.

EXAMPLE 3 Identification of Cell Membrane Associated Molecules asFactors Inhibiting Schwann Cell Migration

This Example illustrates the identification of molecules responsible fordifferential rates of Schwann cell migration.

A. Effects of cell matrix and diffusible factors on Schwann cellmigration

We examined whether the differential rates of Schwann cell migration ondifferent cell types could be due either to secreted molecules, to thedifferent properties of the extracellular matrices, or to cell membraneassociated molecules (i.e., cadherin). In order to determine whethermatrix or secreted molecules were responsible, we assayed Schwann cellmigration on extracellular matrix and in the presence of conditionedmedium. The micro inverted coverslip migration assay was employed.Surfaces laden with astroglial matrix were produced by lysing astrocytesgrown on coverslips with PBS and Triton X-100 (0.1%). Schwanncell-covered fragments were inverted onto the matrix preparations andthe maximum migratory distances of the cells were assessed. Controlexperiments were performed utilizing laminin as substrate. Schwann cellswere found to migrate distances of 0.79 mm±0.02 on astroglial matrix,slightly less than that seen upon laminin (1.09 mm±0.05), but furtherthan on whole cells (0.24 mm±0.03; FIG. 5).

In order to assess the contribution of diffusible factors,astrocyte-conditioned serum free medium (ACM) was used to conductSchwann cell migration assays from laminin. Schwann cells were found tohave migrated distances up to 10.04 mm±0.02 upon laminin in the presenceof ACM whereas migration upon laminin in serum free (SF) medium alonewas 0.59 mm±0.04 (FIG. 6). It appears that pro-migratory factors existin serum and paradoxically ACM.

These experiments suggest that neither astrocyte matrix nor secretedmolecules are inhibitory to Schwann cell migration. The inhibition musttherefore be cell surface mediated.

B Movement of single Schwann cells on laminin and monolayers

The inverted coverslip migration assay described in the previous sectioninvolves a number of different cell interactions, namely Schwanncell-Schwann cell interactions and the adhesion between the Schwanncells and the overlying glass fragment. In order to analyze a simplersituation, time lapse videomicroscopy was used to determine themigration of single Schwann cells on differing cellular andproteinaceous substrates.

Cells were plated onto a 35 mm tissue culture dish and were filmed on aNikon Diaphot inverted microscope mounted in a chamber maintained at 37°C. and at a humidified atmosphere of 10% CO₂ in air. The events wererecorded on a Panasonic 8051 video recorder at 8 frames every 30 secondsfor a period of 14-25 hours. Three types of culture were established:

1. Astrocyte, fibroblast or meningeal monolayers were grown toconfluence within 35 mm tissue culture dishes and filled with 2 ml ofDMEM supplemented with 10% FCS. A 50 μl Schwann cell suspensioncontaining approximately 1000 cells was then added to the dish which wasthen transferred to the timelapse chamber. Filming was initiatedimmediately and Schwann cells were clearly identifiable landing andattaching to the underlying monolayer. Movement of the Schwann cell bodywas recorded every 30 minutes for 6 hour periods by marking the positionof its nucleus onto an acetate sheet covering the monitor. Pathways ofmigration were therefore constructed. Distance moved by the cell bodyevery half hour was measured and used to generate an average speed ofmigration for the cells.

2. Cultures from which interactions between single cells colliding asthey moved on a laminin surface could be filmed were generated asfollows: 1 ml of solution containing 1000 Schwann cells was placed intoa 35 mm culture dish followed by a further 1 ml of an equal number ofeither astroglia or fibroblasts. The dish was transferred to a timelapsechamber and a field of view in which cells of each type were close butnot yet touching was selected. The nature and duration of interactionsbetween the different cell types were recorded.

3. Confrontation assays, in which an expanding monolayer of astrocytesor fibroblasts would come into contact with an expanding monolayer ofSchwann cells, were established using the cell boundaries generated asabove.

Within one such study, single Schwann cells were plated onto astrocytic,fibroblastic or laminin substrates and their cell body movement wasobserved over a time period of 6 hours. The position of the cell bodyafter 30 minute intervals was noted onto an acetate sheet covering themonitor and displacement diagrams were obtained for twenty cells uponeach substrate. From these diagrams, the distance moved every 30 minuteswas obtained and used to generate the average migratory rates of thesingle cells for each of the conditions. A selection of displacementdiagrams are presented as FIG. 5C. It was found that Schwann cellsmigrate the slowest on astrocytes with an average speed of 16.2μm/hr±1.12. They move faster on fibroblasts (31.8 μm/hr±1.39) and attaintheir fastest speed on laminin (64.8 μm/hr±2.88). Therefore, the sametrend as seen with the population migration experiment is seen withsingle Schwann cells. This data is presented graphically as FIG. 7A.

C Interactions between single Schwann cells, astrocytes and fibroblasts

Sparse mixed cultures of Schwann cells and either astrocytes orfibroblasts were established. Regions where single Schwann cells were incontact with isolated astrocytes or fibroblasts were filmed.Astrocyte-Schwann cell (n=50) and fibroblast-Schwann cell (n=40)interactions were observed. When a Schwann cell process encountered anastrocyte, the exploratory growth cone first appeared to attach firmlyto the astrocytic surface and then expand in area, with activelamellipodia exploring the perikarya. The growth cone could be seen tobecome anchored to the astrocyte whilst the cell body would move away,resulting in a very long process connecting the two cells. The averageprocess length was found to be 33.0 μm±3.0 (mean±S.E.M.). In contrastSchwann cells encountering fibroblasts did so via an exploratory growthcone which did not expand on contact. Furthermore, the average processlength between Schwann cells and fibroblasts was found to be 11.8μm±1.85. The longer processes developing between Schwann cells andastrocytes implies greater tension between the cells.

Contacts between Schwann cells and astrocytes were of much longerduration than those between Schwann cells and fibroblasts. Most (80%) ofthe Schwann cell-astrocyte interactions were longer than 90 minutes. Incomparison, only 5% of the Schwann cell-fibroblast interactions were aslong as this. The average length of interaction between Schwann cellsand astrocytes was found to be 257 min±41 minutes whereas the averageSchwann cell-fibroblast-interaction was found to be 48 min±5 (FIG. 7B).A sequence of encounters between a Schwann cell and an astrocytecaptured from a time lapse recording is presented in FIG. 6. Eachconsecutive frame represents a time interval of 2 hours. Thus, Schwanncells appear to interact with astrocytes and fibroblasts differently atthe single cell level. Schwann cells display an exploratory behavior aswell as a static form of interaction with astrocytes. Contact withfibroblasts seems only to involve simple exploration with littleinterruption of Schwann cell migratory movement.

D. Adhesion of Schwann cells to laminin and monolayers

In order to test whether the migratory behavior of Schwann cells ondifferent cell types was a function of adhesivity to the substratum,DiI-labeled Schwann cells were plated onto either astrocytic,fibroblasts, Schwann cell or laminin surfaces. 20,000 DiI-labeledSchwann cells were placed in a 15 mm diameter well in 0.5 ml of mediumover a 13 mm glass coverslip coated with laminin, or a completemonolayer of astrocytes, fibroblasts or Schwann cells and then placedonto a shaking platform (25 rpm) for 30 minutes in an incubator. Thecoverslips were washed three times in Hanks after 30 minutes to removeany non-attached cells and the remainder were fixed for 20 minutes in 4%paraformaldehyde. The number of DiI-labeled Schwann cells that wereattached to the coverslip were counted using a Leitz Diaplan fluorescentmicroscope under rhodamine optics.

The data was normalized by setting Schwann cell adhesion to astrocytesat the arbitrary value of 1±0.03 (mean±S.E.M.). More than twice thenumber of cells adhered to the astrocytic surfaces as compared to eitherthe fibroblastic (0.49±0.02) or laminin surfaces (0.35±0.02). The mostadhesive substrate was found to be Schwann cell monolayers, withadhesion values of 1.58±0.1, compared to astrocytes (FIG. 7). Theanti-migratory astrocyte surface is therefore more adhesive to Schwanncells than are fibroblastic surfaces with in turn are slightly moreadhesive than laminin -coated surfaces. There is therefore an inversecorrelation between rate of Schwann cell migration and adhesion.

The results presented herein show that ACM promoted the migration ofSchwann cells in the absence of serum, and astrocyte matrix is only alittle less good than laminin as a migratory surface. This suggests thatthe majority of the anti-migratory activity displayed by astrocytes isdue to interactions with cell surface associated molecules.

EXAMPLE 4 Effect of Representative Modulation Agents on Schwann CellAdhesion and Migration

The cadherins are known to mediate calcium-dependent cell adhesion(Redies and Takeichi, Dev. Biol 180:413-423, 1996; Munro and Blaschuk,“The Structure, Function and Regulation of Cadherins,” in Cell Adhesionand Cancer Metastasis (P. Brodt ed.) pp. 17-34 (R. G. Landes Co., 1996).Lowering the external calcium to 0.2 mM has been shown to disruptcadherin-mediated interactions between Schwann cells and other celltypes (Letourneau et al., Neurobiol. 22:707-720, 1991). This exampleillustrates the use of calcium or two representative modulating agentsto disrupt cadherin function and increase Schwann cell migration.

A. The Effect of Lowering External Calcium Concentrations on Schwanncell Adhesion and Migration

In order to reduce extracellular calcium, DMEM was replaced by S-MEM(Joklik's modification; Gibco) with 0.2 mM calcium chloride added or thecalcium buffer EGTA (Sigma) was employed. Adhesion assays in the absenceof external calcium were performed using Schwann cells and astrocytes.Either a low calcium solution (S-MEM in place of DMEM with 0.2 mMcalcium chloride) or a calcium buffer (EGTA in a normal DMEM medium) wasused in these assays. Various concentrations of EGTA were tested; theoptimal concentration for Schwann cell migration was found to be 1.6 mM.EGTA concentrations less than 1.3 mM had little effect upon Schwann cellmigration whereas those above 1.8 mM caused disruption of the astrocyticmonolayer (data not shown). Adhesion of Schwann cells to astrocytes inthe presence of the standard DMEM based medium was taken as the controland assigned the normalized value of 1.0±0.03. Low calcium solutionsreduced intercellular adhesion to 0.47±0.09 and the addition of 1.6 mMEGTA to DMEM reduced adhesion to 0.39±0.05 (FIG. 10A). EGTA, being themore effective adhesion inhibitor, was incorporated into the migrationassay and found to increase the extent of Schwann cell migration uponastrocyte monolayers to 0.86 mm±0.06 compared to control migration(0.25±0.03; FIGS. 10B and 10C).

B. The Effect of Representative Modulating Agents on Adhesion andMigration

The following modulating agents were employed at concentrations of 1mg/ml, LRAHAVDING-NH₂ (SEQ ID NO: 21), MRAHAVDING-NH₂ (SEQ ID NO: 23),and the control peptide LRAHGVDING-NH₂ (SEQ ID NO: 30). The former twopeptide modulating agents harbor the cadherin CAR sequence, HAV.Cadherin function was also blocked utilizing the rabbit anti-cadherinCAR sequence antiserum designated as L7 (1:20). Normal rabbit serum(NRS; Sigma, St. Louis, Mo.) and the goat anti-neural cell adhesionmolecule (NCAM) antiserum (Santa Cruz Biotechnology Inc., Santa Cruz,Calif.) were also used at a dilution of 1:20 as controls.

The modulating agents LRAHAVDING-NH₂ (SEQ ID NO: 21) and MRAHAVDING-NH₂(SEQ ID NO: 23) were found to reduce Schwann cell-astrocyte adhesion(0.38±0.07 and 0.39±0.04, respective) as compared to the normalizedadhesion in the absence of peptide (1.0±0.05). The control peptideLRAHGVDING-NH₂ (SEQ ID NO: 30) did not significantly alter Schwanncell-astrocyte adhesion (0.78±0.10; p>0.05; FIG. 11A). Furthermore, therabbit antiserum L7, shown to be specific for the cadherin CAR sequence(Alexander et al., J. Cell Physiol. 156:610-618, 1993) and reported toblock N-cadherin mediated adhesion (Newton et al., Dev. Dynamics197:1-13, 1993) reduced Schwann cell-astrocyte adhesion to 0.39±0.06 ascompared to the normalized control adhesion 1.0±0.09 in the absence ofantibody. This effect was not due to non-specific factors within theantibody sera as rabbit serum had little effect upon intercellularadhesion (0.96±0.05). The control and NCAM antibody also did not affectintracellular adhesion (0.99±0.08).

As a further control, the entire adhesion experiment was repeated usingSchwann cell monolayers as the adhesive substrate, thereby assayingSchwann cell- Schwann cell adhesion. The antiserum L7 was found todisrupt Schwann cell- Schwann cell adhesion to a value of 0.5±0.05compared to the normalized control adhesion 1.0±0.16. The addition ofNRS and the polyclonal NCAM antibody yielded adhesion values of0.96±0.06 and 0.99±0.08, respectively (FIG. 11B). Having shown theability of L7 to disrupt both Schwann cell-astrocyte and Schwanncell-Schwann cell adhesion, the antibody was employed within themigration assay. Schwann cells were found to migrate poorly onastrocytes in the presence of control medium (0.16 mm±0.03), NRS (0.12mm±0.02) or polyclonal anti-NCAM (0.15 mm±0.02). In comparison treatmentof the cultures with the L7 antiserum more than tripled the maximummigration distance of Schwann cells on astrocytes (0.51 mmn±0.04; FIGS.9C, 9D, 10C, and 10D). This effect was not due to disruption of theastroglial monolayer which remained intact (FIG. 10C).

Thus, disrupting cadherin function alters Schwann cell adhesion andmigration. Schwann cells adhere to astrocytes more strongly than tofibroblasts and laminin, and nearly as strongly as to other Schwanncells. In the above experiments, the number of cells adhering toastrocytes was halved by subjecting cultures to calcium withdrawal or bytreating the cells either with modulating agents containing the cadherinCAR sequence or with the L7 antiserum which is directed against the CARsequence. Schwann cell-Schwann cell adhesion was also reduced by L7antiserum. Both calcium withdrawal and the presence of L7 antiserumincreased the rate of Schwann cell migration on astrocytes approximatelythree-fold.

These results demonstrate that modulating agents containing the cadherinCAR sequence and antibodies directed against that sequence are capableof disrupting cadherin function. These results also indicate that themain family of CAMs involved in Schwann cell adhesion and migration arethe cadherins, and that blocking cadherin mediated adhesive interactionsprovides a viable approach for enhancing Schwann cell migration withinthe CNS. Modulating agents capable of interfering with cadherin functionmay be used to facilitate the grafting of Schwann cells into the CNS topromote remyelination of axon regeneration, and for other purposes wherea modulation of cell adhesion is desired.

From the foregoing, it will be evident that although specificembodiments of the invention have been described herein for the purposeof illustrating the invention, various modifications may be made withoutdeviating from the spirit and scope of the invention.

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30 Lys Val Phe Tyr Ser Ile Thr Gly Gln Gly Ala Asp ThrPro Pro Val 35 40 45 Gly Val Phe Ile Ile Glu Arg Glu Thr Gly Trp Leu LysVal Thr Glu 50 55 60 Pro Leu Asp Arg Glu Arg Ile Ala Thr Tyr Thr Leu PheSer His Ala 65 70 75 80 Val Ser Ser Asn Gly Asn Ala Val Glu Asp Pro MetGlu Ile Leu Ile 85 90 95 Thr Val Thr Asp Gln Asn Asp Asn Lys Pro Glu Phe100 105 108 amino acids amino acid linear unknown 9 Asp Trp Val Ile ProPro Ile Ser Cys Pro Glu Asn Glu Lys Gly Glu 1 5 10 15 Phe Pro Lys AsnLeu Val Gln Ile Lys Ser Asn Arg Asp Lys Glu Thr 20 25 30 Lys Val Phe TyrSer Ile Thr Gly Gln Gly Ala Asp Lys Pro Pro Val 35 40 45 Gly Val Phe IleIle Glu Arg Glu Thr Gly Trp Leu Lys Val Thr Gln 50 55 60 Pro Leu Asp ArgGlu Ala Ile Ala Lys Tyr Ile Leu Tyr Ser His Ala 65 70 75 80 Val Ser SerAsn Gly Glu Ala Val Glu Asp Pro Met Glu Ile Val Ile 85 90 95 Thr Val ThrAsp Gln Asn Asp Asn Arg Pro Glu Phe 100 105 6 amino acids amino acidlinear unknown 10 His Ala Val His Ala Val 1 5 13 amino acids amino acidlinear unknown 11 Ser 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What is claimed is:
 1. A method for treating a demyelinatingneurological disease in a mammal, comprising implanting in a mammal: (a)a cell adhesion modulating agent that is 6-50 amino acid residues inlength, wherein said modulating agent comprises the sequence His-Ala-Valand at least one flanking amino acid residue present within anendogenous N-cadherin sequence selected from the group consisting of SEQID NOs:3-5, and wherein said modulating agent inhibitsN-cadherin-mediated cell adhesion; and (b) one or more cells selectedfrom the group consisting of Schwann cells and oligodendrocyteprogenitor cells from individuals not affected with a demyelinatingdisease; wherein said modulating agent and said cell(s) are implantedinto the mammal's central nervous system in an amount sufficient toinhibit N-cadherin-mediated cell adhesion, thereby facilitating saidcell(s) migration and treating a demyelinating neurological disease. 2.A method according to claim 1, wherein said modulating agent comprises asequence selected from the group consisting of LRAHAVDING (SEQ IDNO:21), LRAHAVDVNG (SEQ ID NO:22), MRAHAVDING (SEQ ID NO:23),HLGAHAVDINGNQVET (SEQ ID NO:24), FHLRAHAVDINGNQV (SEQ ID NO:25),LYSHAVSSNG (SEQ ID NO:27), AHAVSE (SEQ ID NO:28) and derivatives of theforegoing sequences comprising one or more modifications selected fromthe group consisting of esterification of the C-terminal carboxylate;amidation of the C-terminal carboxylate; acetylation of the N-terminalamino group; alkoxycarbonylation of the N-terminal amino group; andmethylation, benzylation, t-butylation, tosylation oralkoxycarbonylation of a side chain functional group.
 3. A methodaccording to claim 1, wherein said modulating agent is linked to atargeting agent.
 4. A method according to claim 1, wherein saidmodulating agent is linked to a drug.
 5. A method according to claim 1,wherein said modulating agent further comprises at least one celladhesion recognition sequence bound by an adhesion molecule selectedfrom the group consisting of desmogleins, desmocollins, integrins,N-CAM, occludin, laminin, fibronectin, collagens, vitronectin, entactinand tenascin.
 6. A method according to claim 1, wherein said modulatingagent is administered by implantation with Schwann cells.
 7. A methodaccording to claim 1, wherein said modulating agent is administered byimplantation with oligodendrocyte progenitor cells.
 8. A methodaccording to claim 1, wherein said modulating agent is present within apharmaceutical composition comprising a pharmaceutically acceptablecarrier.
 9. A method according to claim 1, wherein said disease ismultiple sclerosis.
 10. A method according to claim 1, wherein themodulating agent and the cell(s) are administered simultaneously.